Publications

@article{pmid40773290,

title = {A ratiometric catalog of protein isoform shifts in the cardiac fetal gene program},

author = {Yu Han and Shaonil Binti and Sara A Wennersten and Boomathi Pandi and Dominic Cm Ng and Edward Lau and Maggie Py Lam},

doi = {10.1172/jci.insight.184309},

issn = {2379-3708},

year  = {2025},

date = {2025-08-01},

journal = {JCI Insight},

abstract = {Pathological cardiac remodeling is associated with the reactivation of fetal genes, yet the extent of the heart's fetal gene program and its impact on proteome compositions remain incompletely understood. Here, using a new proteome-wide protein ratio quantification strategy with mass spectrometry, we identify pervasive isoform usage shifts in fetal and postnatal mouse hearts, involving 145 pairs of highly homologous paralogs and alternative splicing-derived isoform proteins. Proteome-wide ratio comparisons readily rediscover hallmark fetal gene signatures in muscle contraction and glucose metabolism pathways, while revealing novel isoform usage in mitochondrial and gene expression proteins, including PPA1/PPA2, ANT1/ANT2, and PCBP1/PCBP2 switches. Paralogs with differential fetal usage tend to be evolutionarily recent, consistent with functional diversification. Alternative splicing adds another rich source of fetal isoform usage differences, involving PKM M1/M2, GLS-1 KGA/GAC, PDLIM5 long/short, and other spliceoforms. When comparing absolute protein proportions, we observe a partial reversion toward fetal gene usage in pathological hearts. In summary, we present a ratiometric catalog of paralogs and spliceoform pairs in the cardiac fetal gene program. More generally, the results demonstrate the potential of applying the proteome-wide ratio test concept to discover new regulatory modalities beyond differential gene expression.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39975278,

title = {Deuterium labeling enables proteome wide turnover kinetics analysis in cell culture},

author = {Lorena Alamillo and Dominic C M Ng and Jordan Currie and Alexander Black and Boomathi Pandi and Vyshnavi Manda and Jay Pavelka and Peyton Schaal and Joshua G Travers and Timothy A McKinsey and Maggie P Y Lam and Edward Lau},

doi = {10.1101/2025.01.30.635596},

issn = {2692-8205},

year  = {2025},

date = {2025-01-01},

journal = {bioRxiv},

abstract = {The half-life of proteins is tightly regulated and underlies many cellular processes. It remains unclear the extent to which proteins are dynamically synthesized and degraded in different cell types and cell states. We introduce an improved DO labeling workflow and apply it to examine the landscape of protein turnover in pluripotent and differentiating human induced pluripotent stem cells (hiPSC). The majority of hiPSC proteins show minimal turnover beyond cell doubling rates, but we also identify over 100 new fast-turnover proteins not previously described as short-lived. These include proteins that function in cell division and cell cycle checkpoints, that are enriched in APC/C and SPOP degrons, and that are depleted upon pluripotency exit. Differentiation rapidly shifts the set of fast-turnover proteins toward including RNA binding and splicing proteins. The ability to identify fast-turnover proteins in different cell cultures also facilitates secretome analysis, as exemplified by studies of hiPSC-derived cardiac myocytes and primary human cardiac fibroblasts. The presented workflow is broadly applicable to protein turnover studies in diverse primary, pluripotent, and transformed cells.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40100644,

title = {Improved Method to Determine Protein Turnover Rates with Heavy Water Labeling by Mass Isotopomer Ratio Selection},

author = {Jordan Currie and Dominic C M Ng and Boomathi Pandi and Alexander Black and Vyshnavi Manda and Cheyanne Durham and Jay Pavelka and Maggie P Y Lam and Edward Lau},

doi = {10.1021/acs.jproteome.4c01012},

issn = {1535-3907},

year  = {2025},

date = {2025-04-01},

journal = {J Proteome Res},

volume = {24},

number = {4},

pages = {1992--2005},

abstract = {The synthesis and degradation rates of proteins form an essential component of gene expression control. Heavy water labeling has been used in conjunction with mass spectrometry to measure protein turnover rates, but the optimal analytical approaches to derive turnover rates from the mass isotopomer patterns of deuterium-labeled peptides continue to be a subject of research. Here, we describe a method that comprises (1) a nearest lookup of numerically approximated peptide isotope envelopes, coupled to (2) the selection of optimal mass isotopomer pairs based on peptide sequence rules, to calculate the molar fraction of new peptide synthesis in heavy water labeling mass spectrometry experiments. We validated our approach using an experimental calibration standard comprising mixtures of fully unlabeled and fully labeled proteomes. We then reanalyzed 17 proteome-wide turnover experiments from four mouse organs across multiple data sets and showed that the combined nearest-lookup and rule-based mass isotopomer ratio selection method increases the coverage of well-fitted peptides in protein turnover experiments by up to 58 ± 13%. The workflow is implemented in the Riana software tool for protein turnover analysis and may avail ongoing efforts to study the synthesis and degradation kinetics of proteins in animals on a proteome-wide scale.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40799479,

title = {Protein Turnover Dynamics Analysis With Subcellular Spatial Resolution},

author = {Lorena Alamillo and Alexander Black and Maggie P Y Lam and Edward Lau},

doi = {10.21769/BioProtoc.5409},

issn = {2331-8325},

year  = {2025},

date = {2025-08-01},

journal = {Bio Protoc},

volume = {15},

number = {15},

pages = {e5409},

abstract = {Protein synthesis and degradation (i.e., turnover) forms an important part of protein homeostasis and has been implicated in many age-associated diseases. Different cellular locations, such as organelles and membraneless compartments, often contain individual protein quality control and degradation machineries. Conventional methods to assess protein turnover across subcellular compartments require targeted genetic manipulation or isolation of specific organelles. Here we describe a protocol for simultaneous proteome localization and turnover (SPLAT) analysis, which combines protein turnover measurements with unbiased subcellular spatial proteomics to measure compartment-specific protein turnover rates on a proteome-wide scale. This protocol utilizes dynamic stable isotope labeling of amino acids in cell culture (dynamic SILAC) to resolve the temporal information of protein turnover and multi-step differential ultracentrifugation to assign proteins to multiple subcellular localizations. We further incorporate 2D liquid chromatography fractionation to greatly increase analytical depth while multiplexing with tandem mass tags (TMT) to reduce acquisition time 10-fold. This protocol resolves the spatial and temporal distributions of proteins and can also reveal temporally distinct spatial localizations within a protein pool. Key features • Captures protein turnover rates and subcellular localization of proteins. • Hyperplexing of dynamic SILAC and TMT LOPIT-DC in MS1 and MS2 level data. • Sample collection and processing can be completed within 1 week. • Allows comparison of organellar proteome turnover rates.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38895333,

title = {Improved determination of protein turnover rate with heavy water labeling by mass isotopomer ratio selection},

author = {Jordan Currie and Dominic C M Ng and Boomathi Pandi and Alexander Black and Vyshnavi Manda and Jay Pavelka and Maggie P Y Lam and Edward Lau},

doi = {10.1101/2024.06.04.597043},

issn = {2692-8205},

year  = {2024},

date = {2024-06-01},

journal = {bioRxiv},

abstract = {The synthesis and degradation rates of proteins form an essential component of gene expression control. Heavy water labeling has been used in conjunction with mass spectrometry to measure protein turnover rates, but the optimal analytical approaches to derive turnover rates from the isotopomer patterns of deuterium labeled peptides continue to be a subject of research. Here we describe a method, which comprises a reverse lookup of numerically approximated peptide isotope envelopes, coupled to the selection of optimal isotopomer pairs based on peptide sequence, to calculate the molar fraction of new peptide synthesis in heavy water labeling mass spectrometry experiments. We validated this approach using an experimental calibration curve comprising mixtures of fully unlabeled and fully labeled proteomes. We then re-analyzed 17 proteome-wide turnover experiments from four mouse organs, and showed that the method increases the coverage of well-fitted peptides in protein turnover experiments by 25-82%. The method is implemented in the Riana software tool for protein turnover analysis, and may avail ongoing efforts to study the synthesis and degradation kinetics of proteins in animals on a proteome-wide scale.nnWHAT’S NEW: We describe a reverse lookup method to calculate the molar fraction of new synthesis from numerically approximated peptide isotopomer profiles in heavy water labeling mass spectrometry experiments. Using an experimental calibration curve comprising mixtures of fully unlabeled and fully labeled proteomes at various proportions, we show that this method provides a straightforward way to calculate the proportion of new proteins in a protein pool from arbitrarily chosen isotopomer ratios. We next analyzed which of the isotopomer pairs within the peptide isotope envelope yielded isotopomer time courses that fit most closely to kinetic models, and found that the identity of the isotopomer pair depends partially on the number of deuterium accessible labeling sites of the peptide. We next derived a strategy to automatically select the isotopomer pairs to calculate turnover rates based on peptide sequence, and showed that this increases the coverage of existing proteome-wide turnover experiments in multiple data sets of the mouse heart, liver, kidney, and skeletal muscle by up to 25-82%.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38467653,

title = {Simultaneous proteome localization and turnover analysis reveals spatiotemporal features of protein homeostasis disruptions},

author = {Jordan Currie and Vyshnavi Manda and Sean K Robinson and Celine Lai and Vertica Agnihotri and Veronica Hidalgo and R W Ludwig and Kai Zhang and Jay Pavelka and Zhao V Wang and June-Wha Rhee and Maggie P Y Lam and Edward Lau},

doi = {10.1038/s41467-024-46600-5},

issn = {2041-1723},

year  = {2024},

date = {2024-03-01},

journal = {Nat Commun},

volume = {15},

number = {1},

pages = {2207},

abstract = {The spatial and temporal distributions of proteins are critical to protein function, but cannot be directly assessed by measuring protein bundance. Here we describe a mass spectrometry-based proteomics strategy, Simultaneous Proteome Localization and Turnover (SPLAT), to measure concurrently protein turnover rates and subcellular localization in the same experiment. Applying the method, we find that unfolded protein response (UPR) has different effects on protein turnover dependent on their subcellular location in human AC16 cells, with proteome-wide slowdown but acceleration among stress response proteins in the ER and Golgi. In parallel, UPR triggers broad differential localization of proteins including RNA-binding proteins and amino acid transporters. Moreover, we observe newly synthesized proteins including EGFR that show a differential localization under stress than the existing protein pools, reminiscent of protein trafficking disruptions. We next applied SPLAT to an induced pluripotent stem cell derived cardiomyocyte (iPSC-CM) model of cancer drug cardiotoxicity upon treatment with the proteasome inhibitor carfilzomib. Paradoxically, carfilzomib has little effect on global average protein half-life, but may instead selectively disrupt sarcomere protein homeostasis. This study provides a view into the interactions of protein spatial and temporal dynamics and demonstrates a method to examine protein homeostasis regulations in stress and drug response.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid36711879,

title = {Simultaneous proteome localization and turnover analysis reveals spatiotemporal features of protein homeostasis disruptions},

author = {Jordan Currie and Vyshnavi Manda and Sean K Robinson and Celine Lai and Vertica Agnihotri and Veronica Hidalgo and R W Ludwig and Kai Zhang and Jay Pavelka and Zhao V Wang and June-Wha Rhee and Maggie P Y Lam and Edward Lau},

doi = {10.1101/2023.01.04.521821},

issn = {2692-8205},

year  = {2024},

date = {2024-01-01},

journal = {bioRxiv},

abstract = {The functions of proteins depend on their spatial and temporal distributions, which are not directly measured by static protein abundance. Under endoplasmic reticulum (ER) stress, the unfolded protein response (UPR) pathway remediates proteostasis in part by altering the turnover kinetics and spatial distribution of proteins. A global view of these spatiotemporal changes has yet to emerge and it is unknown how they affect different cellular compartments and pathways. Here we describe a mass spectrometry-based proteomics strategy and data analysis pipeline, termed Simultaneous Proteome Localization and Turnover (SPLAT), to measure concurrently the changes in protein turnover and subcellular distribution in the same experiment. Investigating two common UPR models of thapsigargin and tunicamycin challenge in human AC16 cells, we find that the changes in protein turnover kinetics during UPR varies across subcellular localizations, with overall slowdown but an acceleration in endoplasmic reticulum and Golgi proteins involved in stress response. In parallel, the spatial proteomics component of the experiment revealed an externalization of amino acid transporters and ion channels under UPR, as well as the migration of RNA-binding proteins toward an endosome co-sedimenting compartment. The SPLAT experimental design classifies heavy and light SILAC labeled proteins separately, allowing the observation of differential localization of new and old protein pools and capturing a partition of newly synthesized EGFR and ITGAV to the ER under stress that suggests protein trafficking disruptions. Finally, application of SPLAT toward human induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) exposed to the cancer drug carfilzomib, identified a selective disruption of proteostasis in sarcomeric proteins as a potential mechanism of carfilzomib-mediated cardiotoxicity. Taken together, this study provides a global view into the spatiotemporal dynamics of human cardiac cells and demonstrates a method for inferring the coordinations between spatial and temporal proteome regulations in stress and drug response.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38456420,

title = {Tissue Usage Preference and Intrinsically Disordered Region Remodeling of Alternative Splicing Derived Proteoforms in the Heart},

author = {Boomathi Pandi and Stella Brenman and Alexander Black and Dominic C M Ng and Edward Lau and Maggie P Y Lam},

doi = {10.1021/acs.jproteome.3c00789},

issn = {1535-3907},

year  = {2024},

date = {2024-08-01},

journal = {J Proteome Res},

volume = {23},

number = {8},

pages = {3161--3173},

abstract = {A computational analysis of mass spectrometry data was performed to uncover alternative splicing derived protein variants across chambers of the human heart. Evidence for 216 non-canonical isoforms was apparent in the atrium and the ventricle, including 52 isoforms not documented on SwissProt and recovered using an RNA sequencing derived database. Among non-canonical isoforms, 29 show signs of regulation based on statistically significant preferences in tissue usage, including a ventricular enriched protein isoform of tensin-1 (TNS1) and an atrium-enriched PDZ and LIM Domain 3 (PDLIM3) isoform 2 (PDLIM3-2/ALP-H). Examined variant regions that differ between alternative and canonical isoforms are highly enriched with intrinsically disordered regions. Moreover, over two-thirds of such regions are predicted to function in protein binding and RNA binding. The analysis here lends further credence to the notion that alternative splicing diversifies the proteome by rewiring intrinsically disordered regions, which are increasingly recognized to play important roles in the generation of biological function from protein sequences.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35575093,

title = {HDAC6 modulates myofibril stiffness and diastolic function of the heart},

author = {Ying-Hsi Lin and Jennifer L Major and Tim Liebner and Zaynab Hourani and Joshua G Travers and Sara A Wennersten and Korey R Haefner and Maria A Cavasin and Cortney E Wilson and Mark Y Jeong and Yu Han and Michael Gotthardt and Scott K Ferguson and Amrut V Ambardekar and Maggie Py Lam and Chunaram Choudhary and Henk L Granzier and Kathleen C Woulfe and Timothy A McKinsey},

doi = {10.1172/JCI148333},

issn = {1558-8238},

year  = {2022},

date = {2022-05-01},

journal = {J Clin Invest},

volume = {132},

number = {10},

abstract = {Passive stiffness of the heart is determined largely by extracellular matrix and titin, which functions as a molecular spring within sarcomeres. Titin stiffening is associated with the development of diastolic dysfunction (DD), while augmented titin compliance appears to impair systolic performance in dilated cardiomyopathy. We found that myofibril stiffness was elevated in mice lacking histone deacetylase 6 (HDAC6). Cultured adult murine ventricular myocytes treated with a selective HDAC6 inhibitor also exhibited increased myofibril stiffness. Conversely, HDAC6 overexpression in cardiomyocytes led to decreased myofibril stiffness, as did ex vivo treatment of mouse, rat, and human myofibrils with recombinant HDAC6. Modulation of myofibril stiffness by HDAC6 was dependent on 282 amino acids encompassing a portion of the PEVK element of titin. HDAC6 colocalized with Z-disks, and proteomics analysis suggested that HDAC6 functions as a sarcomeric protein deacetylase. Finally, increased myofibril stiffness in HDAC6-deficient mice was associated with exacerbated DD in response to hypertension or aging. These findings define a role for a deacetylase in the control of myofibril function and myocardial passive stiffness, suggest that reversible acetylation alters titin compliance, and reveal the potential of targeting HDAC6 to manipulate the elastic properties of the heart to treat cardiac diseases.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid36356032,

title = {Protein prediction models support widespread post-transcriptional regulation of protein abundance by interacting partners},

author = {Himangi Srivastava and Michael J Lippincott and Jordan Currie and Robert Canfield and Maggie P Y Lam and Edward Lau},

doi = {10.1371/journal.pcbi.1010702},

issn = {1553-7358},

year  = {2022},

date = {2022-11-01},

journal = {PLoS Comput Biol},

volume = {18},

number = {11},

pages = {e1010702},

abstract = {Protein and mRNA levels correlate only moderately. The availability of proteogenomics data sets with protein and transcript measurements from matching samples is providing new opportunities to assess the degree to which protein levels in a system can be predicted from mRNA information. Here we examined the contributions of input features in protein abundance prediction models. Using large proteogenomics data from 8 cancer types within the Clinical Proteomic Tumor Analysis Consortium (CPTAC) data set, we trained models to predict the abundance of over 13,000 proteins using matching transcriptome data from up to 958 tumor or normal adjacent tissue samples each, and compared predictive performances across algorithms, data set sizes, and input features. Over one-third of proteins (4,648) showed relatively poor predictability (elastic net r ≤ 0.3) from their cognate transcripts. Moreover, we found widespread occurrences where the abundance of a protein is considerably less well explained by its own cognate transcript level than that of one or more trans locus transcripts. The incorporation of additional trans-locus transcript abundance data as input features increasingly improved the ability to predict sample protein abundance. Transcripts that contribute to non-cognate protein abundance primarily involve those encoding known or predicted interaction partners of the protein of interest, including not only large multi-protein complexes as previously shown, but also small stable complexes in the proteome with only one or few stable interacting partners. Network analysis further shows a complex proteome-wide interdependency of protein abundance on the transcript levels of multiple interacting partners. The predictive model analysis here therefore supports that protein-protein interaction including in small protein complexes exert post-transcriptional influence on proteome compositions more broadly than previously recognized. Moreover, the results suggest mRNA and protein co-expression analysis may have utility for finding gene interactions and predicting expression changes in biological systems.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35930447,

title = {Proteogenomics reveals sex-biased aging genes and coordinated splicing in cardiac aging},

author = {Yu Han and Sara A Wennersten and Julianna M Wright and R W Ludwig and Edward Lau and Maggie P Y Lam},

doi = {10.1152/ajpheart.00244.2022},

issn = {1522-1539},

year  = {2022},

date = {2022-09-01},

journal = {Am J Physiol Heart Circ Physiol},

volume = {323},

number = {3},

pages = {H538--H558},

abstract = {The risks of heart diseases are significantly modulated by age and sex, but how these factors influence baseline cardiac gene expression remains incompletely understood. Here, we used RNA sequencing and mass spectrometry to compare gene expression in female and male young adult (4 mo) and early aging (20 mo) mouse hearts, identifying thousands of age- and sex-dependent gene expression signatures. Sexually dimorphic cardiac genes are broadly distributed, functioning in mitochondrial metabolism, translation, and other processes. In parallel, we found over 800 genes with differential aging response between male and female, including genes in cAMP and PKA signaling. Analysis of the sex-adjusted aging cardiac transcriptome revealed a widespread remodeling of exon usage patterns that is largely independent from differential gene expression, concomitant with upstream changes in RNA-binding protein and splice factor transcripts. To evaluate the impact of the splicing events on cardiac proteoform composition, we applied an RNA-guided proteomics computational pipeline to analyze the mass spectrometry data and detected hundreds of putative splice variant proteins that have the potential to rewire the cardiac proteome. Taken together, the results here suggest that cardiac aging is associated with 1) widespread sex-biased aging genes and 2) a rewiring of RNA splicing programs, including sex- and age-dependent changes in exon usages and splice patterns that have the potential to influence cardiac protein structure and function. These changes contribute to the emerging evidence for considerable sexual dimorphism in the cardiac aging process that should be considered in the search for disease mechanisms. Han et al. used proteogenomics to compare male and female mouse hearts at 4 and 20 mo. Sex-biased cardiac genes function in mitochondrial metabolism, translation, autophagy, and other processes. Hundreds of cardiac genes show sex-by-age interactions, that is, sex-biased aging genes. Cardiac aging is accompanied with a remodeling of exon usage in functionally coordinated genes, concomitant with differential expression of RNA-binding proteins and splice factors. These features represent an underinvestigated aspect of cardiac aging that may be relevant to the search for disease mechanisms.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33549679,

title = {Computation-assisted targeted proteomics of alternative splicing protein isoforms in the human heart},

author = {Yu Han and Silas D Wood and Julianna M Wright and Vishantie Dostal and Edward Lau and Maggie P Y Lam},

doi = {10.1016/j.yjmcc.2021.01.007},

issn = {1095-8584},

year  = {2021},

date = {2021-05-01},

journal = {J Mol Cell Cardiol},

volume = {154},

pages = {92--96},

abstract = {Alternative splicing is prevalent in the heart and implicated in many cardiovascular diseases, but not every alternative transcript is translated and detecting non-canonical isoforms at the protein level remains challenging. Here we show the use of a computation-assisted targeted proteomics workflow to detect protein alternative isoforms in the human heart. We build on a recent strategy to integrate deep RNA-seq and large-scale mass spectrometry data to identify candidate translated isoform peptides. A machine learning approach is then applied to predict their fragmentation patterns and design protein isoform-specific parallel reaction monitoring detection (PRM) assays. As proof-of-principle, we built PRM assays for 29 non-canonical isoform peptides and detected 22 peptides in a human heart lysate. The predictions-aided PRM assays closely mirrored synthetic peptide standards for non-canonical sequences. This approach may be useful for validating non-canonical protein identification and discovering functionally relevant isoforms in the heart.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34779440,

title = {Shotgun Proteomics Sample Processing Automated by an Open-Source Lab Robot},

author = {Yu Han and Cody T Thomas and Sara A Wennersten and Edward Lau and Maggie P Y Lam},

doi = {10.3791/63092},

issn = {1940-087X},

year  = {2021},

date = {2021-10-01},

journal = {J Vis Exp},

number = {176},

abstract = {Mass spectrometry-based shotgun proteomics experiments require multiple sample preparation steps, including enzymatic protein digestion and clean-up, which can take up significant person-hours of bench labor and present a source of batch-to-batch variability. Lab automation with pipetting robots can reduce manual work, maximize throughput, and increase research reproducibility. Still, the steep starting prices of standard automation stations make them unaffordable for many academic laboratories. This article describes a proteomics sample preparation workflow using an affordable, open-source automation system (The Opentrons OT-2), including instructions for setting up semi-automated protein reduction, alkylation, digestion, and clean-up steps; as well as accompanying open-source Python scripts to program the OT-2 system through its application programming interface.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33067450,

title = {A high-stringency blueprint of the human proteome},

author = {Subash Adhikari and Edouard C Nice and Eric W Deutsch and Lydie Lane and Gilbert S Omenn and Stephen R Pennington and Young-Ki Paik and Christopher M Overall and Fernando J Corrales and Ileana M Cristea and Jennifer E Van Eyk and Mathias Uhlén and Cecilia Lindskog and Daniel W Chan and Amos Bairoch and James C Waddington and Joshua L Justice and Joshua LaBaer and Henry Rodriguez and Fuchu He and Markus Kostrzewa and Peipei Ping and Rebekah L Gundry and Peter Stewart and Sanjeeva Srivastava and Sudhir Srivastava and Fabio C S Nogueira and Gilberto B Domont and Yves Vandenbrouck and Maggie P Y Lam and Sara Wennersten and Juan Antonio Vizcaino and Marc Wilkins and Jochen M Schwenk and Emma Lundberg and Nuno Bandeira and Gyorgy Marko-Varga and Susan T Weintraub and Charles Pineau and Ulrike Kusebauch and Robert L Moritz and Seong Beom Ahn and Magnus Palmblad and Michael P Snyder and Ruedi Aebersold and Mark S Baker},

doi = {10.1038/s41467-020-19045-9},

issn = {2041-1723},

year  = {2020},

date = {2020-10-01},

journal = {Nat Commun},

volume = {11},

number = {1},

pages = {5301},

abstract = {The Human Proteome Organization (HUPO) launched the Human Proteome Project (HPP) in 2010, creating an international framework for global collaboration, data sharing, quality assurance and enhancing accurate annotation of the genome-encoded proteome. During the subsequent decade, the HPP established collaborations, developed guidelines and metrics, and undertook reanalysis of previously deposited community data, continuously increasing the coverage of the human proteome. On the occasion of the HPP's tenth anniversary, we here report a 90.4% complete high-stringency human proteome blueprint. This knowledge is essential for discerning molecular processes in health and disease, as we demonstrate by highlighting potential roles the human proteome plays in our understanding, diagnosis and treatment of cancers, cardiovascular and infectious diseases.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33377032,

title = {Determining Alternative Protein Isoform Expression Using RNA Sequencing and Mass Spectrometry},

author = {Yu Han and Julianna M Wright and Edward Lau and Maggie Pui Yu Lam},

doi = {10.1016/j.xpro.2020.100138},

issn = {2666-1667},

year  = {2020},

date = {2020-12-01},

journal = {STAR Protoc},

volume = {1},

number = {3},

pages = {100138},

abstract = {Alternative splicing greatly expands the coding capacity of the human genome, but how many alternative transcripts are translated as proteins or carry functional importance remains unknown and awaits experimental verification. Here, we describe a protocol that combines transcriptomics (RNA-seq) and proteomics (mass spectrometry [MS]) analyses to identify alternative isoforms in proteomes. This workflow is applicable to custom-generated RNA-seq and MS data from matching samples, as well as the reanalysis of existing transcriptomics and proteomics datasets in public repositories. For complete details on the use and execution of this protocol, please refer to Lau et al. (2019).},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33116222,

title = {Proteomic signatures of acute oxidative stress response to paraquat in the mouse heart},

author = {Vishantie Dostal and Silas D Wood and Cody T Thomas and Yu Han and Edward Lau and Maggie P Y Lam},

doi = {10.1038/s41598-020-75505-8},

issn = {2045-2322},

year  = {2020},

date = {2020-10-01},

journal = {Sci Rep},

volume = {10},

number = {1},

pages = {18440},

abstract = {The heart is sensitive to oxidative damage but a global view on how the cardiac proteome responds to oxidative stressors has yet to fully emerge. Using quantitative tandem mass spectrometry, we assessed the effects of acute exposure of the oxidative stress inducer paraquat on protein expression in mouse hearts. We observed widespread protein expression changes in the paraquat-exposed heart especially in organelle-containing subcellular fractions. During cardiac response to acute oxidative stress, proteome changes are consistent with a rapid reduction of mitochondrial metabolism, coupled with activation of multiple antioxidant proteins, reduction of protein synthesis and remediation of proteostasis. In addition to differential expression, we saw evidence of spatial reorganizations of the cardiac proteome including the translocation of hexokinase 2 to more soluble fractions. Treatment with the antioxidants Tempol and MitoTEMPO reversed many proteomic signatures of paraquat but this reversal was incomplete. We also identified a number of proteins with unknown function in the heart to be triggered by paraquat, suggesting they may have functions in oxidative stress response. Surprisingly, protein expression changes in the heart correlate poorly with those in the lung, consistent with differential sensitivity or stress response in these two organs. The results and data set here could provide insights into oxidative stress responses in the heart and avail the search for new therapeutic targets.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31409188,

title = {Dynamic Chromatin Targeting of BRD4 Stimulates Cardiac Fibroblast Activation},

author = {Matthew S Stratton and Rushita A Bagchi and Marina B Felisbino and Rachel A Hirsch and Harrison E Smith and Andrew S Riching and Blake Y Enyart and Keith A Koch and Maria A Cavasin and Michael Alexanian and Kunhua Song and Jun Qi and Madeleine E Lemieux and Deepak Srivastava and Maggie P Y Lam and Saptarsi M Haldar and Charles Y Lin and Timothy A McKinsey},

doi = {10.1161/CIRCRESAHA.119.315125},

issn = {1524-4571},

year  = {2019},

date = {2019-09-01},

journal = {Circ Res},

volume = {125},

number = {7},

pages = {662--677},

abstract = {RATIONALE: Small molecule inhibitors of the acetyl-histone binding protein BRD4 have been shown to block cardiac fibrosis in preclinical models of heart failure (HF). However, since the inhibitors target BRD4 ubiquitously, it is unclear whether this chromatin reader protein functions in cell type-specific manner to control pathological myocardial fibrosis. Furthermore, the molecular mechanisms by which BRD4 stimulates the transcriptional program for cardiac fibrosis remain unknown.nnOBJECTIVE: We sought to test the hypothesis that BRD4 functions in a cell-autonomous and signal-responsive manner to control activation of cardiac fibroblasts, which are the major extracellular matrix-producing cells of the heart.nnMETHODS AND RESULTS: RNA-sequencing, mass spectrometry, and cell-based assays employing primary adult rat ventricular fibroblasts demonstrated that BRD4 functions as an effector of TGF-β (transforming growth factor-β) signaling to stimulate conversion of quiescent cardiac fibroblasts into  ()-positive cells that express high levels of extracellular matrix. These findings were confirmed in vivo through whole-transcriptome analysis of cardiac fibroblasts from mice subjected to transverse aortic constriction and treated with the small molecule BRD4 inhibitor, JQ1. Chromatin immunoprecipitation-sequencing revealed that BRD4 undergoes stimulus-dependent, genome-wide redistribution in cardiac fibroblasts, becoming enriched on a subset of enhancers and super-enhancers, and leading to RNA polymerase II activation and expression of downstream target genes. Employing the  (SERTA domain-containing protein 4) locus as a prototype, we demonstrate that dynamic chromatin targeting of BRD4 is controlled, in part, by p38 MAPK (mitogen-activated protein kinase) and provide evidence of a critical function for  in TGF-β-mediated cardiac fibroblast activation.nnCONCLUSIONS: These findings define BRD4 as a central regulator of the pro-fibrotic cardiac fibroblast phenotype, establish a p38-dependent signaling circuit for epigenetic reprogramming in heart failure, and uncover a novel role for . The work provides a mechanistic foundation for the development of BRD4 inhibitors as targeted anti-fibrotic therapies for the heart.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31599600,

title = {Mining the Proteome Associated with Rheumatic and Autoimmune Diseases},

author = {Cristina Ruiz-Romero and Maggie P Y Lam and Peter Nilsson and Patrik Önnerfjord and Paul J Utz and Jennifer E Van Eyk and Vidya Venkatraman and Justyna Fert-Bober and Fiona E Watt and Francisco J Blanco},

doi = {10.1021/acs.jproteome.9b00360},

issn = {1535-3907},

year  = {2019},

date = {2019-12-01},

journal = {J Proteome Res},

volume = {18},

number = {12},

pages = {4231--4239},

abstract = {A steady increase in the incidence of osteoarthritis and other rheumatic diseases has been observed in recent decades, including autoimmune conditions such as rheumatoid arthritis, spondyloarthropathies, systemic lupus erythematosus, systemic sclerosis, and Sjögren's syndrome. Rheumatic and autoimmune diseases (RADs) are characterized by the inflammation of joints, muscles, or other connective tissues. In addition to often experiencing debilitating mobility and pain, RAD patients are also at a higher risk of suffering comorbidities such as cardiovascular or infectious events. Given the socioeconomic impact of RADs, broad research efforts have been dedicated to these diseases worldwide. In the present work, we applied literature mining platforms to identify "popular" proteins closely related to RADs. The platform is based on publicly available literature. The results not only will enable the systematic prioritization of candidates to perform targeted proteomics studies but also may lead to a greater insight into the key pathogenic processes of these disorders.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31825849,

title = {Splice-Junction-Based Mapping of Alternative Isoforms in the Human Proteome},

author = {Edward Lau and Yu Han and Damon R Williams and Cody T Thomas and Rajani Shrestha and Joseph C Wu and Maggie P Y Lam},

doi = {10.1016/j.celrep.2019.11.026},

issn = {2211-1247},

year  = {2019},

date = {2019-12-01},

journal = {Cell Rep},

volume = {29},

number = {11},

pages = {3751--3765.e5},

abstract = {The protein-level translational status and function of many alternative splicing events remain poorly understood. We use an RNA sequencing (RNA-seq)-guided proteomics method to identify protein alternative splicing isoforms in the human proteome by constructing tissue-specific protein databases that prioritize transcript splice junction pairs with high translational potential. Using the custom databases to reanalyze ∼80 million mass spectra in public proteomics datasets, we identify more than 1,500 noncanonical protein isoforms across 12 human tissues, including ∼400 sequences undocumented on TrEMBL and RefSeq databases. We apply the method to original quantitative mass spectrometry experiments and observe widespread isoform regulation during human induced pluripotent stem cell cardiomyocyte differentiation. On a proteome scale, alternative isoform regions overlap frequently with disordered sequences and post-translational modification sites, suggesting that alternative splicing may regulate protein function through modulating intrinsically disordered regions. The described approach may help elucidate functional consequences of alternative splicing and expand the scope of proteomics investigations in various systems.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30256117,

title = {Identifying High-Priority Proteins Across the Human Diseasome Using Semantic Similarity},

author = {Edward Lau and Vidya Venkatraman and Cody T Thomas and Joseph C Wu and Jennifer E Van Eyk and Maggie P Y Lam},

doi = {10.1021/acs.jproteome.8b00393},

issn = {1535-3907},

year  = {2018},

date = {2018-12-01},

journal = {J Proteome Res},

volume = {17},

number = {12},

pages = {4267--4278},

abstract = {Identifying the genes and proteins associated with a biological process or disease is a central goal of the biomedical research enterprise. However, relatively few systematic approaches are available that provide objective evaluation of the genes or proteins known to be important to a research topic, and hence researchers often rely on subjective evaluation of domain experts and laborious manual literature review. Computational bibliometric analysis, in conjunction with text mining and data curation, attempts to automate this process and return prioritized proteins in any given research topic. We describe here a method to identify and rank protein-topic relationships by calculating the semantic similarity between a protein and a query term in the biomerical literature while adjusting for the impact and immediacy of associated research articles. We term the calculated metric the weighted copublication distance (WCD) and show that it compares well to related approaches in predicting benchmark protein lists in multiple biological processes. We used WCD to extract prioritized "popular proteins" across multiple cell types, subanatomical regions, and standardized vocabularies containing over 20 000 human disease terms. The collection of protein-disease associations across the resulting human "diseasome" supports data analytical workflows to perform reverse protein-to-disease queries and functional annotation of experimental protein lists. We envision that the described improvement to the popular proteins strategy will be useful for annotating protein lists and guiding method development efforts as well as generating new hypotheses on understudied disease proteins using bibliometric information.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29317621,

title = {Integrated omics dissection of proteome dynamics during cardiac remodeling},

author = {Edward Lau and Quan Cao and Maggie P Y Lam and Jie Wang and Dominic C M Ng and Brian J Bleakley and Jessica M Lee and David A Liem and Ding Wang and Henning Hermjakob and Peipei Ping},

doi = {10.1038/s41467-017-02467-3},

issn = {2041-1723},

year  = {2018},

date = {2018-01-01},

journal = {Nat Commun},

volume = {9},

number = {1},

pages = {120},

abstract = {Transcript abundance and protein abundance show modest correlation in many biological models, but how this impacts disease signature discovery in omics experiments is rarely explored. Here we report an integrated omics approach, incorporating measurements of transcript abundance, protein abundance, and protein turnover to map the landscape of proteome remodeling in a mouse model of pathological cardiac hypertrophy. Analyzing the hypertrophy signatures that are reproducibly discovered from each omics data type across six genetic strains of mice, we find that the integration of transcript abundance, protein abundance, and protein turnover data leads to 75% gain in discovered disease gene candidates. Moreover, the inclusion of protein turnover measurements allows discovery of post-transcriptional regulations across diverse pathways, and implicates distinct disease proteins not found in steady-state transcript and protein abundance data. Our results suggest that multi-omics investigations of proteome dynamics provide important insights into disease pathogenesis in vivo.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26977904,

title = {A large dataset of protein dynamics in the mammalian heart proteome},

author = {Edward Lau and Quan Cao and Dominic C M Ng and Brian J Bleakley and T Umut Dincer and Brian M Bot and Ding Wang and David A Liem and Maggie P Y Lam and Junbo Ge and Peipei Ping},

doi = {10.1038/sdata.2016.15},

issn = {2052-4463},

year  = {2016},

date = {2016-03-01},

journal = {Sci Data},

volume = {3},

pages = {160015},

abstract = {Protein stability is a major regulatory principle of protein function and cellular homeostasis. Despite limited understanding on mechanisms, disruption of protein turnover is widely implicated in diverse pathologies from heart failure to neurodegenerations. Information on global protein dynamics therefore has the potential to expand the depth and scope of disease phenotyping and therapeutic strategies. Using an integrated platform of metabolic labeling, high-resolution mass spectrometry and computational analysis, we report here a comprehensive dataset of the in vivo half-life of 3,228 and the expression of 8,064 cardiac proteins, quantified under healthy and hypertrophic conditions across six mouse genetic strains commonly employed in biomedical research. We anticipate these data will aid in understanding key mitochondrial and metabolic pathways in heart diseases, and further serve as a reference for methodology development in dynamics studies in multiple organ systems.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27980500,

title = {Cardiovascular proteomics in the era of big data: experimental and computational advances},

author = {Maggie P Y Lam and Edward Lau and Dominic C M Ng and Ding Wang and Peipei Ping},

doi = {10.1186/s12014-016-9124-y},

issn = {1542-6416},

year  = {2016},

date = {2016-01-01},

journal = {Clin Proteomics},

volume = {13},

pages = {23},

abstract = {Proteomics plays an increasingly important role in our quest to understand cardiovascular biology. Fueled by analytical and computational advances in the past decade, proteomics applications can now go beyond merely inventorying protein species, and address sophisticated questions on cardiac physiology. The advent of massive mass spectrometry datasets has in turn led to increasing intersection between proteomics and big data science. Here we review new frontiers in technological developments and their applications to cardiovascular medicine. The impact of big data science on cardiovascular proteomics investigations and translation to medicine is highlighted.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27356587,

title = {Data-Driven Approach To Determine Popular Proteins for Targeted Proteomics Translation of Six Organ Systems},

author = {Maggie P Y Lam and Vidya Venkatraman and Yi Xing and Edward Lau and Quan Cao and Dominic C M Ng and Andrew I Su and Junbo Ge and Jennifer E Van Eyk and Peipei Ping},

doi = {10.1021/acs.jproteome.6b00095},

issn = {1535-3907},

year  = {2016},

date = {2016-11-01},

journal = {J Proteome Res},

volume = {15},

number = {11},

pages = {4126--4134},

abstract = {Amidst the proteomes of human tissues lie subsets of proteins that are closely involved in conserved pathophysiological processes. Much of biomedical research concerns interrogating disease signature proteins and defining their roles in disease mechanisms. With advances in proteomics technologies, it is now feasible to develop targeted proteomics assays that can accurately quantify protein abundance as well as their post-translational modifications; however, with rapidly accumulating number of studies implicating proteins in diseases, current resources are insufficient to target every protein without judiciously prioritizing the proteins with high significance and impact for assay development. We describe here a data science method to prioritize and expedite assay development on high-impact proteins across research fields by leveraging the biomedical literature record to rank and normalize proteins that are popularly and preferentially published by biomedical researchers. We demonstrate this method by finding priority proteins across six major physiological systems (cardiovascular, cerebral, hepatic, renal, pulmonary, and intestinal). The described method is data-driven and builds upon the collective knowledge of previous publications referenced on PubMed to lend objectivity to target selection. The method and resulting popular protein lists may also be useful for exploring biological processes associated with various physiological systems and research topics, in addition to benefiting ongoing efforts to facilitate the broad translation of proteomics technologies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26463451,

title = {Fibrotic-like changes in degenerate human intervertebral discs revealed by quantitative proteomic analysis},

author = {A Yee and M P Y Lam and V Tam and W C W Chan and I K Chu and K S E Cheah and K M C Cheung and D Chan},

doi = {10.1016/j.joca.2015.09.020},

issn = {1522-9653},

year  = {2016},

date = {2016-03-01},

journal = {Osteoarthritis Cartilage},

volume = {24},

number = {3},

pages = {503--513},

abstract = {OBJECTIVE: Intervertebral disc degeneration (IDD) can lead to symptomatic conditions including sciatica and back pain. The purpose of this study is to understand the extracellular matrix (ECM) changes in disc biology through comparative proteomic analysis of degenerated and non-degenerated human intervertebral disc (IVD) tissues of different ages.nnDESIGN: Seven non-degenerated (11-46 years of age) and seven degenerated (16-53 years of age) annulus fibrosus (AF) and nucleus pulposus (NP) samples were used. Proteins were extracted using guanidine hydrochloride, separated from large proteoglycans (PGs) by caesium chloride (CsCl) density gradient ultracentrifugation, and identified using liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). For quantitative comparison, proteins were labeled with iTRAQ reagents. Collagen fibrils in the NP were assessed using scanning electron microscopy (SEM).nnRESULTS: In the AF, quantitative analysis revealed increased levels of HTRA1, COMP and CILP in degeneration when compared with samples from older individuals. Fibronectin showed increment with age and degeneration. In the NP, more CILP and CILP2 were present in degenerated samples of younger individuals. Reduced protein solubility was observed in degenerated and older non-degenerated samples correlated with an accumulation of type I collagen in the insoluble fibers. Characterization of collagen fibrils in the NP revealed smaller mean fibril diameters and decreased porosity in the degenerated samples.nnCONCLUSIONS: Our study identified distinct matrix changes associated with aging and degeneration in the intervertebral discs (IVDs). The nature of the ECM changes, together with observed decreased in solubility and changes in fibril diameter is consistent with a fibrotic-like environment.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28007144,

title = {Proteomics Research in Cardiovascular Medicine and Biomarker Discovery},

author = {Maggie P Y Lam and Peipei Ping and Elizabeth Murphy},

doi = {10.1016/j.jacc.2016.10.031},

issn = {1558-3597},

year  = {2016},

date = {2016-12-01},

journal = {J Am Coll Cardiol},

volume = {68},

number = {25},

pages = {2819--2830},

abstract = {Proteomics is a systems physiology discipline to address the large-scale characterization of protein species within a biological system, be it a cell, a tissue, a body biofluid, an organism, or a cohort population. Building on advances from chemical analytical platforms (e.g., mass spectrometry and other technologies), proteomics approaches have contributed powerful applications in cardiovascular biomedicine, most notably in: 1) the discovery of circulating protein biomarkers of heart diseases from plasma samples; and 2) the identification of disease mechanisms and potential therapeutic targets in cardiovascular tissues, in both preclinical models and translational studies. Contemporary proteomics investigations offer powerful means to simultaneously examine tens of thousands of proteins in various samples, and understand their molecular phenotypes in health and disease. This concise review introduces study design considerations, example applications and use cases, as well as interpretation and analysis of proteomics data in cardiovascular biomedicine.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26821878,

title = {The contribution of Alu exons to the human proteome},

author = {Lan Lin and Peng Jiang and Juw Won Park and Jinkai Wang and Zhi-Xiang Lu and Maggie P Y Lam and Peipei Ping and Yi Xing},

doi = {10.1186/s13059-016-0876-5},

issn = {1474-760X},

year  = {2016},

date = {2016-01-01},

journal = {Genome Biol},

volume = {17},

pages = {15},

abstract = {BACKGROUND: Alu elements are major contributors to lineage-specific new exons in primate and human genomes. Recent studies indicate that some Alu exons have high transcript inclusion levels or tissue-specific splicing profiles, and may play important regulatory roles in modulating mRNA degradation or translational efficiency. However, the contribution of Alu exons to the human proteome remains unclear and controversial. The prevailing view is that exons derived from young repetitive elements, such as Alu elements, are restricted to regulatory functions and have not had adequate evolutionary time to be incorporated into stable, functional proteins.nnRESULTS: We adopt a proteotranscriptomics approach to systematically assess the contribution of Alu exons to the human proteome. Using RNA sequencing, ribosome profiling, and proteomics data from human tissues and cell lines, we provide evidence for the translational activities of Alu exons and the presence of Alu exon derived peptides in human proteins. These Alu exon peptides represent species-specific protein differences between primates and other mammals, and in certain instances between humans and closely related primates. In the case of the RNA editing enzyme ADARB1, which contains an Alu exon peptide in its catalytic domain, RNA sequencing analyses of A-to-I editing demonstrate that both the Alu exon skipping and inclusion isoforms encode active enzymes. The Alu exon derived peptide may fine tune the overall editing activity and, in limited cases, the site selectivity of ADARB1 protein products.nnCONCLUSIONS: Our data indicate that Alu elements have contributed to the acquisition of novel protein sequences during primate and human evolution.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26123431,

title = {Priorities and trends in the study of proteins in eye research, 1924-2014},

author = {Richard D Semba and Maggie Lam and Kai Sun and Pingbo Zhang and Debra A Schaumberg and Luigi Ferrucci and Peipei Ping and Jennifer E Van Eyk},

doi = {10.1002/prca.201500006},

issn = {1862-8354},

year  = {2015},

date = {2015-12-01},

journal = {Proteomics Clin Appl},

volume = {9},

number = {11-12},

pages = {1105--1122},

abstract = {PURPOSE: To identify the proteins that are relevant to eye research and develop assays for the study of a set of these proteins.nnEXPERIMENTAL DESIGN: We conducted a bibliometric analysis by merging gene lists for human and mouse from the National Center for Biotechnology Information FTP site and combining them with PubMed references that were retrieved with the search terms "eye" [MeSH Terms] OR "eye" [All Fields] OR "eyes" [All Fields].nnRESULTS: For human and mouse eye studies, respectively, the total number of publications was 13,525 and 23,895 and the total number of proteins was 4050 and 4717. For proteins in human and mouse eye studies, respectively, 88.7 and 81.7% had five or fewer citations. The top 50 most intensively studied proteins for human and mouse eye studies were generally in the areas of photoreceptors and phototransduction, inflammation, and angiogenesis, neurodevelopment, lens transparency, and cell-cycle and cellular processes. We proposed selected reaction monitoring assays that were developed in silico for the top fifty most intensively studied proteins in human and mouse eye research.nnCONCLUSIONS AND CLINICAL RELEVANCE: We conclude that scientists engaged in eye research tend to focus on the same proteins. Newer resources and tools in proteomics can expand the investigations to lesser-known proteins of the eye.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid25752359,

title = {Spatial and temporal dynamics of the cardiac mitochondrial proteome},

author = {Edward Lau and Derrick Huang and Quan Cao and T Umut Dincer and Caitie M Black and Amanda J Lin and Jessica M Lee and Ding Wang and David A Liem and Maggie P Y Lam and Peipei Ping},

doi = {10.1586/14789450.2015.1024227},

issn = {1744-8387},

year  = {2015},

date = {2015-04-01},

journal = {Expert Rev Proteomics},

volume = {12},

number = {2},

pages = {133--146},

abstract = {Mitochondrial proteins alter in their composition and quantity drastically through time and space in correspondence to changing energy demands and cellular signaling events. The integrity and permutations of this dynamism are increasingly recognized to impact the functions of the cardiac proteome in health and disease. This article provides an overview on recent advances in defining the spatial and temporal dynamics of mitochondrial proteins in the heart. Proteomics techniques to characterize dynamics on a proteome scale are reviewed and the physiological consequences of altered mitochondrial protein dynamics are discussed. Lastly, we offer our perspectives on the unmet challenges in translating mitochondrial dynamics markers into the clinic.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid24946186,

title = {Characterization of human plasma proteome dynamics using deuterium oxide},

author = {Ding Wang and David A Liem and Edward Lau and Dominic C M Ng and Brian J Bleakley and Martin Cadeiras and Mario C Deng and Maggie P Y Lam and Peipei Ping},

doi = {10.1002/prca.201400038},

issn = {1862-8354},

year  = {2014},

date = {2014-08-01},

journal = {Proteomics Clin Appl},

volume = {8},

number = {7-8},

pages = {610--619},

abstract = {PURPOSE: High-throughput quantification of human protein turnover via in vivo administration of deuterium oxide ((2) H2 O) is a powerful new approach to examine potential disease mechanisms. Its immediate clinical translation is contingent upon characterizations of the safety and hemodynamic effects of in vivo administration of (2) H2 O to human subjects.nnEXPERIMENTAL DESIGN: We recruited ten healthy human subjects with a broad demographic variety to evaluate the safety, feasibility, efficacy, and reproducibility of (2) H2 O intake for studying protein dynamics. We designed a protocol where each subject orally consumed weight-adjusted doses of 70% (2) H2 O daily for 14 days to enrich body water and proteins with deuterium. Plasma proteome dynamics was measured using a high-resolution MS method we recently developed.nnRESULTS: This protocol was successfully applied in ten human subjects to characterize the endogenous turnover rates of 542 human plasma proteins, the largest such human dataset to-date. Throughout the study, we did not detect physiological effects or signs of discomfort from (2) H2 O consumption.nnCONCLUSIONS AND CLINICAL RELEVANCE: Our investigation supports the utility of a (2) H2 O intake protocol that is safe, accessible, and effective for clinical investigations of large-scale human protein turnover dynamics. This workflow shows promising clinical translational value for examining plasma protein dynamics in human diseases.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid24791980,

title = {Fully automatable multidimensional reversed-phase liquid chromatography with online tandem mass spectrometry},

author = {Maggie P Y Lam and Chun Hin Law and Quan Quan and Yun Zhao and Ivan K Chu},

doi = {10.1007/978-1-4939-0685-7_3},

issn = {1940-6029},

year  = {2014},

date = {2014-01-01},

journal = {Methods Mol Biol},

volume = {1156},

pages = {39--51},

abstract = {Liquid chromatography (LC) is essential for sample fractionation in shotgun proteomics applications. With suitable design, common LC separation chemistries, including reversed-phase (RP) and strong cation exchange (SCX) mode, can be combined in online multidimensional LC to greatly enhance the overall separation power and, thus, proteome coverage. This protocol describes the design and assembly of a flexible online multidimensional RP-SCX-RP LC system that is compatible with deep proteome profiling on common shotgun proteomics platforms.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid24957502,

title = {Lysine ubiquitination and acetylation of human cardiac 20S proteasomes},

author = {Nobel Zong and Peipei Ping and Edward Lau and Howard Jh Choi and Dominic Cm Ng and David Meyer and Caiyun Fang and Haomin Li and Ding Wang and Ivette M Zelaya and John R Yates and Maggie Py Lam},

doi = {10.1002/prca.201400029},

issn = {1862-8354},

year  = {2014},

date = {2014-08-01},

journal = {Proteomics Clin Appl},

volume = {8},

number = {7-8},

pages = {590--594},

abstract = {PURPOSE: Altered proteasome functions are associated with multiple cardiomyopathies. While the proteasome targets polyubiquitinated proteins for destruction, it itself is modifiable by ubiquitination. We aim to identify the exact ubiquitination sites on cardiac proteasomes and examine whether they are also subject to acetylations.nnEXPERIMENTAL DESIGN: Assembled cardiac 20S proteasome complexes were purified from five human hearts with ischemic cardiomyopathy, then analyzed by high-resolution MS to identify ubiquitination and acetylation sites. We developed a library search strategy that may be used to complement database search in identifying PTM in different samples.nnRESULTS: We identified 63 ubiquitinated lysines from intact human cardiac 20S proteasomes. In parallel, 65 acetylated residues were also discovered, 39 of which shared with ubiquitination sites.nnCONCLUSION AND CLINICAL RELEVANCE: This is the most comprehensive characterization of cardiac proteasome ubiquitination to date. There are significant overlaps between the discovered ubiquitination and acetylation sites, permitting potential crosstalk in regulating proteasome functions. The information presented here will aid future therapeutic strategies aimed at regulating the functions of cardiac proteasomes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid24614109,

title = {Protein kinetic signatures of the remodeling heart following isoproterenol stimulation},

author = {Maggie P Y Lam and Ding Wang and Edward Lau and David A Liem and Allen K Kim and Dominic C M Ng and Xiangbo Liang and Brian J Bleakley and Chenguang Liu and Jason D Tabaraki and Martin Cadeiras and Yibin Wang and Mario C Deng and Peipei Ping},

doi = {10.1172/JCI73787},

issn = {1558-8238},

year  = {2014},

date = {2014-04-01},

journal = {J Clin Invest},

volume = {124},

number = {4},

pages = {1734--1744},

abstract = {Protein temporal dynamics play a critical role in time-dimensional pathophysiological processes, including the gradual cardiac remodeling that occurs in early-stage heart failure. Methods for quantitative assessments of protein kinetics are lacking, and despite knowledge gained from single-protein studies, integrative views of the coordinated behavior of multiple proteins in cardiac remodeling are scarce. Here, we developed a workflow that integrates deuterium oxide (2H2O) labeling, high-resolution mass spectrometry (MS), and custom computational methods to systematically interrogate in vivo protein turnover. Using this workflow, we characterized the in vivo turnover kinetics of 2,964 proteins in a mouse model of β-adrenergic-induced cardiac remodeling. The data provided a quantitative and longitudinal view of cardiac remodeling at the molecular level, revealing widespread kinetic regulations in calcium signaling, metabolism, proteostasis, and mitochondrial dynamics. We translated the workflow to human studies, creating a reference dataset of 496 plasma protein turnover rates from 4 healthy adults. The approach is applicable to short, minimal label enrichment and can be performed on as little as a single biopsy, thereby overcoming critical obstacles to clinical investigations. The protein turnover quantitation experiments and computational workflow described here should be widely applicable to large-scale biomolecular investigations of human disease mechanisms with a temporal perspective.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid23391412,

title = {A novel spectral library workflow to enhance protein identifications},

author = {Haomin Li and Nobel C Zong and Xiangbo Liang and Allen K Kim and Jeong Ho Choi and Ning Deng and Ivette Zelaya and Maggie Lam and Huilong Duan and Peipei Ping},

doi = {10.1016/j.jprot.2013.01.026},

issn = {1876-7737},

year  = {2013},

date = {2013-04-01},

journal = {J Proteomics},

volume = {81},

pages = {173--184},

abstract = {The innovations in mass spectrometry-based investigations in proteome biology enable systematic characterization of molecular details in pathophysiological phenotypes. However, the process of delineating large-scale raw proteomic datasets into a biological context requires high-throughput data acquisition and processing. A spectral library search engine makes use of previously annotated experimental spectra as references for subsequent spectral analyses. This workflow delivers many advantages, including elevated analytical efficiency and specificity as well as reduced demands in computational capacity. In this study, we created a spectral matching engine to address challenges commonly associated with a library search workflow. Particularly, an improved sliding dot product algorithm, that is robust to systematic drifts of mass measurement in spectra, is introduced. Furthermore, a noise management protocol distinguishes spectra correlation attributed from noise and peptide fragments. It enables elevated separation between target spectral matches and false matches, thereby suppressing the possibility of propagating inaccurate peptide annotations from library spectra to query spectra. Moreover, preservation of original spectra also accommodates user contributions to further enhance the quality of the library. Collectively, this search engine supports reproducible data analyses using curated references, thereby broadening the accessibility of proteomics resources to biomedical investigators. This article is part of a Special Issue entitled: From protein structures to clinical applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid23242529,

title = {Adrenomedullin enhances invasion of human extravillous cytotrophoblast-derived cell lines by regulation of urokinase plasminogen activator expression and s-nitrosylation},

author = {Ben S T Wong and Kevin K W Lam and Cheuk-Lun Lee and Vera H H Wong and Maggie P Y Lam and Ivan K Chu and William S B Yeung and Philip C N Chiu},

doi = {10.1095/biolreprod.112.103903},

issn = {1529-7268},

year  = {2013},

date = {2013-02-01},

journal = {Biol Reprod},

volume = {88},

number = {2},

pages = {34},

abstract = {Extravillous cytotrophoblast (EVCT) is responsible for trophoblast invasion, which is an important process during placentation. Dysregulation of the process is associated with a wide range of pregnancy complications. Adrenomedullin (ADM) is a polypeptide expressed most abundantly in first-trimester placentas. We hypothesized that ADM modulated the invasion of human EVCT. Our results showed that ADM enhanced invasion and migration but not proliferation in two EVCT cell lines, JEG-3 and TEV-1. Similar observation can also be obtained in primary EVCTs. JEG-3 and TEV-1 cells expressed ADM receptor components as demonstrated by immunostaining, Western blotting, and RT-PCR. The ADM antagonist ADM(22-52) (ADM C-terminal 22-52 amino acid fragment) suppressed ADM-induced invasion and migration, confirming that ADM exerted its biological effects through its classical receptors. The stimulatory effect of ADM on EVCT invasiveness was associated with induction (P < 0.05) of urokinase plasminogen activator (uPA) and nitric oxide synthase (NOS) expression and activity. Silencing of uPA by siRNA transfection abolished the stimulatory effect of ADM, suggesting that uPA is the key mediator for ADM-induced invasion. The involvement of NO in enhancing the invasion and biosynthesis of uPA in EVCT cell lines was confirmed by using pharmacological inhibitors of NOS and NO donors. ADM-mediated NO production also increased protein S-nitrosylation of JEG-3 cells. S-nitrosylation activated uPA in vitro and induced a higher proteinase activity. These findings provide indications that ADM and its downstream NO signaling may play an important role in modulating human EVCT functions.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid24014600,

title = {Identification of CD147 (basigin) as a mediator of trophoblast functions},

author = {Cheuk-Lun Lee and Maggie P Y Lam and Kevin K W Lam and Carmen O N Leung and Ronald T K Pang and Ivan K Chu and Tiffany H L Wan and Joyce Chai and William S B Yeung and Philip C N Chiu},

doi = {10.1093/humrep/det355},

issn = {1460-2350},

year  = {2013},

date = {2013-11-01},

journal = {Hum Reprod},

volume = {28},

number = {11},

pages = {2920--2929},

abstract = {STUDY QUESTION: Does CD147 regulate trophoblast functions in vitro?nnSUMMARY ANSWER: CD147 exists as a receptor complex on human trophoblast and regulates the implantation, invasion and differentiation of trophoblast.nnWHAT IS KNOWN ALREADY: CD147 is a membrane protein implicated in a variety of physiological and pathological conditions due to its regulation of cell-cell recognition, cell differentiation and tissue remodeling. Reduced placental CD147 expression is associated with pre-eclampsia, but the mechanism of actions remains unclear.nnSTUDY DESIGN, SIZE, DURATION: A loss of function approach or functional blocking antibody was used to study the function of CD147 in primary human cytotrophoblasts isolated from first trimester termination of pregnancy and/or in the BeWo cell line, which possesses characteristics of human cytotrophoblasts.nnPARTICIPANTS/MATERIALS, SETTING METHODS: CD147 expression was analyzed by immunofluorescence staining and western blotting. CD147-associated protein complex on plasma membrane were separated by blue native gel electrophoresis and identified by reversed-phase liquid chromatography coupled with quadrupole time-of-flight hybrid mass spectrometer. Cell proliferation and invasion were determined by fluorometric cell proliferation assays and transwell invasion assays, respectively. Matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) activities were measured by gelatin gel zymography and uPA assay kits, respectively. Cell migration was determined by wound-healing assays. Cell fusion was analyzed by immunocytochemistry staining of E-cadherin and 4',6-diamidino-2-phenylindole. The transcripts of matrix proteinases and trophoblast lineage markers were measured by quantitative PCR. Extracellular signal-regulated kinase (ERK) activation was analyzed by western blot using antibodies against ERKs.nnMAIN RESULTS AND THE ROLE OF CHANCE: CD147 exists as protein complexes on the plasma membrane of primary human cytotrophoblasts and BeWo cells. Several known CD147-interacting partners, including integrin β1 and monocarboxylate transporter-1, were identified. Suppression of CD147 by siRNA significantly (P < 0.05) reduced trophoblast-endometrial cell interaction, cell invasion, syncytialization, differentiation and ERK activation of BeWo cells. Consistently, anti-CD147 functional blocking antibody suppressed the invasiveness of primary human cytotrophoblasts. The reduced invasiveness was probably due to the restrained (P < 0.05) enzyme activities of MMP-2, MMP-9 and uPA.nnLIMITATIONS, REASONS FOR CAUTION: Most of the above findings are based on BeWo cell lines. These results need to be confirmed with human first trimester primary cytotrophoblast.nnWIDER IMPLICATIONS OF THE FINDINGS: This is the first study on the role of CD147 in trophoblast function. Further investigation on the function of CD147 and its associated protein complexes will enhance our understanding on human placentation.nnSTUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by the University of Hong Kong Grant 201011159200. The authors have no competing interests to declare.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid23965338,

title = {Integration of cardiac proteome biology and medicine by a specialized knowledgebase},

author = {Nobel C Zong and Haomin Li and Hua Li and Maggie P Y Lam and Rafael C Jimenez and Christina S Kim and Ning Deng and Allen K Kim and Jeong Ho Choi and Ivette Zelaya and David Liem and David Meyer and Jacob Odeberg and Caiyun Fang and Hao-Jie Lu and Tao Xu and James Weiss and Huilong Duan and Mathias Uhlen and John R Yates and Rolf Apweiler and Junbo Ge and Henning Hermjakob and Peipei Ping},

doi = {10.1161/CIRCRESAHA.113.301151},

issn = {1524-4571},

year  = {2013},

date = {2013-10-01},

journal = {Circ Res},

volume = {113},

number = {9},

pages = {1043--1053},

abstract = {RATIONALE: Omics sciences enable a systems-level perspective in characterizing cardiovascular biology. Integration of diverse proteomics data via a computational strategy will catalyze the assembly of contextualized knowledge, foster discoveries through multidisciplinary investigations, and minimize unnecessary redundancy in research efforts.nnOBJECTIVE: The goal of this project is to develop a consolidated cardiac proteome knowledgebase with novel bioinformatics pipeline and Web portals, thereby serving as a new resource to advance cardiovascular biology and medicine.nnMETHODS AND RESULTS: We created Cardiac Organellar Protein Atlas Knowledgebase (COPaKB; www.HeartProteome.org), a centralized platform of high-quality cardiac proteomic data, bioinformatics tools, and relevant cardiovascular phenotypes. Currently, COPaKB features 8 organellar modules, comprising 4203 LC-MS/MS experiments from human, mouse, drosophila, and Caenorhabditis elegans, as well as expression images of 10,924 proteins in human myocardium. In addition, the Java-coded bioinformatics tools provided by COPaKB enable cardiovascular investigators in all disciplines to retrieve and analyze pertinent organellar protein properties of interest.nnCONCLUSIONS: COPaKB provides an innovative and interactive resource that connects research interests with the new biological discoveries in protein sciences. With an array of intuitive tools in this unified Web server, nonproteomics investigators can conveniently collaborate with proteomics specialists to dissect the molecular signatures of cardiovascular phenotypes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid23022582,

title = {Site-specific quantitative analysis of cardiac mitochondrial protein phosphorylation},

author = {Maggie P Y Lam and Edward Lau and Sarah B Scruggs and Ding Wang and Tae-Young Kim and David A Liem and Jun Zhang and Christopher M Ryan and Kym F Faull and Peipei Ping},

doi = {10.1016/j.jprot.2012.09.015},

issn = {1876-7737},

year  = {2013},

date = {2013-04-01},

journal = {J Proteomics},

volume = {81},

pages = {15--23},

abstract = {We report the development of a multiple-reaction monitoring (MRM) strategy specifically tailored to the detection and quantification of mitochondrial protein phosphorylation. We recently derived 68 MRM transitions specific to protein modifications in the respiratory chain, voltage-dependent anion channel, and adenine nucleotide translocase. Here, we have now expanded the total number of MRM transitions to 176 to cover proteins from the tricarboxylic acid cycle, pyruvate dehydrogenase complex, and branched-chain alpha-keto acid dehydrogenase complex. We utilized the transition set to analyze endogenous protein phosphorylation in human heart, mouse heart, and mouse liver. The data demonstrate the potential utility of the MRM workflow for studying the functional details of mitochondrial phosphorylation signaling. This article is part of a Special Issue entitled: From protein structures to clinical applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid22387130,

title = {An MRM-based workflow for quantifying cardiac mitochondrial protein phosphorylation in murine and human tissue},

author = {Maggie P Y Lam and Sarah B Scruggs and Tae-Young Kim and Chenggong Zong and Edward Lau and Ding Wang and Christopher M Ryan and Kym F Faull and Peipei Ping},

doi = {10.1016/j.jprot.2012.02.014},

issn = {1876-7737},

year  = {2012},

date = {2012-08-01},

journal = {J Proteomics},

volume = {75},

number = {15},

pages = {4602--4609},

abstract = {The regulation of mitochondrial function is essential for cardiomyocyte adaptation to cellular stress. While it has long been understood that phosphorylation regulates flux through metabolic pathways, novel phosphorylation sites are continually being discovered in all functionally distinct areas of the mitochondrial proteome. Extracting biologically meaningful information from these phosphorylation sites requires an adaptable, sensitive, specific and robust method for their quantification. Here we report a multiple reaction monitoring-based mass spectrometric workflow for quantifying site-specific phosphorylation of mitochondrial proteins. Specifically, chromatographic and mass spectrometric conditions for 68 transitions derived from 23 murine and human phosphopeptides, and their corresponding unmodified peptides, were optimized. These methods enabled the quantification of endogenous phosphopeptides from the outer mitochondrial membrane protein VDAC, and the inner membrane proteins ANT and ETC complexes I, III and V. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of mitochondrial protein phosphorylation in cardiac physiology and pathophysiology. This article is part of a Special Issue entitled: Translational Proteomics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid22807358,

title = {Fully automatable two-dimensional hydrophilic interaction liquid chromatography-reversed phase liquid chromatography with online tandem mass spectrometry for shotgun proteomics},

author = {Yun Zhao and Ricky P W Kong and Guohui Li and Maggie P Y Lam and C H Law and Simon M Y Lee and Herman C Lam and Ivan K Chu},

doi = {10.1002/jssc.201200054},

issn = {1615-9314},

year  = {2012},

date = {2012-07-01},

journal = {J Sep Sci},

volume = {35},

number = {14},

pages = {1755--1763},

abstract = {We have developed a fully automatable two-dimensional liquid chromatography platform for shotgun proteomics analyses based on the online coupling of hydrophilic interaction liquid chromatography (HILIC) - using a nonionic type of TSKgel Amide 80 at either pH 6.8 (neutral) or 2.7 (acidic) - with conventional low-pH reversed-phase chromatography. Online coupling of the neutral-pH HILIC and reversed phase chromatography systems outperformed the acidic HILIC-reversed phase chromatography combination, resulting in 18.4% (1914 versus 1617 nonredundant proteins) and 41.6% (12,989 versus 9172 unique peptides) increases in the number of identified peptides and proteins from duplicate analyses of Rat pheochromocytoma lysates. Armed with this optimized HILIC-reversed phase liquid chromatography platform, we identified 2554 nonredundant proteins from duplicate analyses of a Saccharomyces cerevisiae lysate, with the detected protein abundances spanning from approximately 41 to 10(6) copies per cell, which contained up to approximately 2092 different validated protein species with a dynamic range of concentrations of up to approximately 10(4) . This present study establishes a fully automated platform as a promising methodology to enable online coupling of different hydrophilic HILIC and reversed phase chromatography systems, thereby expanding the repertoire of multidimensional liquid chromatography for shotgun proteomics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid22539426,

title = {HUPO 2011: The new Cardiovascular Initiative - integrating proteomics and cardiovascular biology in health and disease},

author = {Maggie P Y Lam and Fernando Vivanco and Arjen Scholten and Henning Hermjakob and Jennifer Van Eyk and Peipei Ping},

doi = {10.1002/pmic.201270015},

issn = {1615-9861},

year  = {2012},

date = {2012-03-01},

journal = {Proteomics},

volume = {12},

number = {6},

pages = {749--751},

abstract = {A newly reorganized HUPO Cardiovascular Initiative was announced at the HUPO 2011 Cardiovascular Initiative Workshop at Geneva. The new initiative is now part of the biology- and disease-driven component of the HUPO Human Proteome Project (B/D-HPP). Here we report the recent achievements and future directions of the initiative, and offer a perspective on the present challenges of cardiovascular proteomics and its integration with the cardiovascular biology community at large.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid22915825,

title = {Metabolic labeling reveals proteome dynamics of mouse mitochondria},

author = {Tae-Young Kim and Ding Wang and Allen K Kim and Edward Lau and Amanda J Lin and David A Liem and Jun Zhang and Nobel C Zong and Maggie P Y Lam and Peipei Ping},

doi = {10.1074/mcp.M112.021162},

issn = {1535-9484},

year  = {2012},

date = {2012-12-01},

journal = {Mol Cell Proteomics},

volume = {11},

number = {12},

pages = {1586--1594},

abstract = {Mitochondrial dysfunction is associated with many human diseases. Mitochondrial damage is exacerbated by inadequate protein quality control and often further contributes to pathogenesis. The maintenance of mitochondrial functions requires a delicate balance of continuous protein synthesis and degradation, i.e. protein turnover. To understand mitochondrial protein dynamics in vivo, we designed a metabolic heavy water ((2)H(2)O) labeling strategy customized to examine individual protein turnover in the mitochondria in a systematic fashion. Mice were fed with (2)H(2)O at a minimal level (<5% body water) without physiological impacts. Mitochondrial proteins were analyzed from 9 mice at each of the 13 time points between 0 and 90 days (d) of labeling. A novel multiparameter fitting approach computationally determined the normalized peak areas of peptide mass isotopomers at initial and steady-state time points and permitted the protein half-life to be determined without plateau-level (2)H incorporation. We characterized the turnover rates of 458 proteins in mouse cardiac and hepatic mitochondria and found median turnover rates of 0.0402 d(-1) and 0.163 d(-1), respectively, corresponding to median half-lives of 17.2 d and 4.26 d. Mitochondria in the heart and those in the liver exhibited distinct turnover kinetics, with limited synchronization within functional clusters. We observed considerable interprotein differences in turnover rates in both organs, with half-lives spanning from hours to months (≈ 60 d). Our proteomics platform demonstrates the first large-scale analysis of mitochondrial protein turnover rates in vivo, with potential applications in translational research.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid22641634,

title = {Perspectives on: SGP symposium on mitochondrial physiology and medicine: mitochondrial proteome design: from molecular identity to pathophysiological regulation},

author = {Jun Zhang and Amanda Lin and Jared Powers and Maggie P Lam and Christopher Lotz and David Liem and Edward Lau and Ding Wang and Ning Deng and Paavo Korge and Nobel C Zong and Hua Cai and James Weiss and Peipei Ping},

doi = {10.1085/jgp.201210797},

issn = {1540-7748},

year  = {2012},

date = {2012-06-01},

journal = {J Gen Physiol},

volume = {139},

number = {6},

pages = {395--406},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid22456183,

title = {Substrate- and isoform-specific proteome stability in normal and stressed cardiac mitochondria},

author = {Edward Lau and Ding Wang and Jun Zhang and Hongxiu Yu and Maggie P Y Lam and Xiangbo Liang and Nobel Zong and Tae-Young Kim and Peipei Ping},

doi = {10.1161/CIRCRESAHA.112.268359},

issn = {1524-4571},

year  = {2012},

date = {2012-04-01},

journal = {Circ Res},

volume = {110},

number = {9},

pages = {1174--1178},

abstract = {RATIONALE: Mitochondrial protein homeostasis is an essential component of the functions and oxidative stress responses of the heart.nnOBJECTIVE: To determine the specificity and efficiency of proteome turnover of the cardiac mitochondria by endogenous and exogenous proteolytic mechanisms.nnMETHODS AND RESULTS: Proteolytic degradation of the murine cardiac mitochondria was assessed by 2-dimensional differential gel electrophoresis and liquid chromatography-tandem mass spectrometry. Mitochondrial proteases demonstrated a substrate preference for basic protein variants, which indicates a possible recognition mechanism based on protein modifications. Endogenous mitochondrial proteases and the cytosolic 20S proteasome exhibited different substrate specificities.nnCONCLUSIONS: The cardiac mitochondrial proteome contains low amounts of proteases and is remarkably stable in isolation. Oxidative damage lowers the proteolytic capacity of cardiac mitochondria and reduces substrate availability for mitochondrial proteases. The 20S proteasome preferentially degrades specific substrates in the mitochondria and may contribute to cardiac mitochondrial proteostasis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid21350782,

title = {Combinatorial use of offline SCX and online RP-RP liquid chromatography for iTRAQ-based quantitative proteomics applications},

author = {Edward Lau and Maggie P Y Lam and S O Siu and Ricky P W Kong and Wai Lung Chan and Zhongjun Zhou and Jirong Huang and Clive Lo and Ivan K Chu},

doi = {10.1039/c1mb05010a},

issn = {1742-2051},

year  = {2011},

date = {2011-05-01},

journal = {Mol Biosyst},

volume = {7},

number = {5},

pages = {1399--1408},

abstract = {Extensive front-end separation is usually required for complex samples in bottom-up proteomics to alleviate the problem of peptide undersampling. Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-based experiments have particularly higher demands, in terms of the number of duty cycles and the sensitivity, to confidently quantify protein abundance. Strong cation exchange (SCX)/reverse phase (RP) liquid chromatography (LC) is currently used routinely to separate iTRAQ-labeled peptides because of its ability to simultaneously clean up the iTRAQ reagents and byproducts and provide first-dimension separation; nevertheless, the low resolution of SCX means that peptides can be redundantly sampled across fractions, leading to loss of usable duty cycles. In this study, we explored the combinatorial application of offline SCX fractionation with online RP-RP applied to iTRAQ-labeled chloroplast proteins to evaluate the effect of three-dimensional LC separation on the overall performance of the quantitative proteomics experiment. We found that the higher resolution of RP-RP can be harnessed to complement SCX-RP and increase the quality of protein identification and quantification, without significantly impacting instrument time and reproducibility.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid21548098,

title = {Fully automatable two-dimensional reversed-phase capillary liquid chromatography with online tandem mass spectrometry for shotgun proteomics},

author = {S O Siu and Maggie P Y Lam and Edward Lau and Ricky P W Kong and Simon M Y Lee and Ivan K Chu},

doi = {10.1002/pmic.201100110},

issn = {1615-9861},

year  = {2011},

date = {2011-06-01},

journal = {Proteomics},

volume = {11},

number = {11},

pages = {2308--2319},

abstract = {Herein, we describe the development of a fully automatable technology that features online coupling of high-pH RP separation with conventional low-pH RP separation in a two-dimensional capillary liquid chromatography (2-D LC) system for shotgun proteomics analyses. The complete analysis comprises 13 separation cycles, each involving transfer of the eluate from the first-dimension, high-pH RP separation onto the second RP dimension for further separation. The solvent strength increases across the 13 fractions (cycles) to elute all peptides for further resolution on the second-dimension, low-pH RP separation, each under identical gradient-elution conditions. The total run time per analysis is 52 h. In triplicate analyses of a lysate of mouse embryonic fibroblasts, we used this technology to identify 2431 non-redundant proteins, of which 50% were observed in all three replicates. A comparison of RP-RP 2-D LC and strong cation exchange-RP 2-D LC analyses reveals that the two technologies identify primarily different peptides, thereby underscoring the differences in their separation chemistries.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid22009802,

title = {Online combination of reversed-phase/reversed-phase and porous graphitic carbon liquid chromatography for multicomponent separation of proteomics and glycoproteomics samples},

author = {Maggie P Y Lam and Edward Lau and S O Siu and Dominic C M Ng and Ricky P W Kong and Philip C N Chiu and William S B Yeung and Clive Lo and Ivan K Chu},

doi = {10.1002/elps.201100092},

issn = {1522-2683},

year  = {2011},

date = {2011-11-01},

journal = {Electrophoresis},

volume = {32},

number = {21},

pages = {2930--2940},

abstract = {In this paper, we describe an online combination of reversed-phase/reversed-phase (RP-RP) and porous graphitic carbon (PGC) liquid chromatography (LC) for multicomponent analysis of proteomics and glycoproteomics samples. The online RP-RP portion of this system provides comprehensive 2-D peptide separation based on sequence hydrophobicity at pH 2 and 10. Hydrophilic components (e.g. glycans, glycopeptides) that are not retained by RP are automatically diverted downstream to a PGC column for further trapping and separation. Furthermore, the RP-RP/PGC system can provide simultaneous extension of the hydropathy range and peak capacity for analysis. Using an 11-protein mixture, we found that the system could efficiently separate native peptides and released N-glycans from a single sample. We evaluated the applicability of the system to the analysis of complex biological samples using 25 μg of the lysate of a human choriocarcinoma cell line (BeWo), confidently identifying a total of 1449 proteins from a single experiment and up to 1909 distinct proteins from technical triplicates. The PGC fraction increased the sequence coverage through the inclusion of additional hydrophilic sequences that accounted for up to 6.9% of the total identified peptides from the BeWo lysate, with apparent preference for the detection of hydrophilic motifs and proteins. In addition, RP-RP/PGC is applicable to the analysis of complex glycomics samples, as demonstrated by our analysis of a concanavalin A-extracted glycoproteome from human serum; in total, 134 potentially N-glycosylated serum proteins, 151 possible N-glycosylation sites, and more than 40 possible N-glycan structures recognized by concanavalin A were simultaneously detected.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid19882125,

title = {N-linked glycoprotein analysis using dual-extraction ultrahigh-performance liquid chromatography and electrospray tandem mass spectrometry},

author = {S O Siu and Maggie P Y Lam and Edward Lau and William S B Yeung and David M Cox and Ivan K Chu},

doi = {10.1007/978-1-60761-454-8_9},

issn = {1940-6029},

year  = {2010},

date = {2010-01-01},

journal = {Methods Mol Biol},

volume = {600},

pages = {133--143},

abstract = {Although reverse-phase liquid chromatography (RP-LC) is a common technique for peptide separation in shotgun proteomics and glycoproteomics, it often provides unsatisfactory results for the analysis of glycopeptides and glycans. This bias against glycopeptides makes it difficult to study glycoproteins. By coupling mass spectrometry (MS) with a combination of RP-LC and normal-phase (NP)-LC as an integrated front-end separation system, we demonstrate that effective identification and characterization of both peptides and glycopeptides mixtures, and their constituent glycan structures, can be achieved from a single sample injection event.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid20632160,

title = {Online coupling of reverse-phase and hydrophilic interaction liquid chromatography for protein and glycoprotein characterization},

author = {Maggie P Y Lam and S O Siu and Edward Lau and Xiuli Mao and H Z Sun and Philip C N Chiu and William S B Yeung and David M Cox and Ivan K Chu},

doi = {10.1007/s00216-010-3991-2},

issn = {1618-2650},

year  = {2010},

date = {2010-09-01},

journal = {Anal Bioanal Chem},

volume = {398},

number = {2},

pages = {791--804},

abstract = {We have developed a novel system for coupling reverse-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) online in a micro-flow scheme. In this approach, the inherent solvent incompatibility between RP and HILIC is overcome through the use of constant-pressure online solvent mixing, which allows our system to perform efficient separations of both hydrophilic and hydrophobic compounds for mass spectrometry-based proteomics applications. When analyzing the tryptic digests of bovine serum albumin, ribonuclease B, and horseradish peroxidase, we observed near-identical coverage of peptides and glycopeptides when using online RP-HILIC--with only a single sample injection event--as we did from two separate RP and HILIC analyses. The coupled system was also capable of concurrently characterizing the peptide and glycan portions of deglycosylated glycoproteins from one injection event, as confirmed, for example, through our detection of 23 novel glycans from turkey ovalbumin. Finally, we validated the applicability of using RP-HILIC for the analysis of highly complex biological samples (mouse chondrocyte lysate, deglycosylated human serum). The enhanced coverage and efficiency of online RP-HILIC makes it a viable technique for the comprehensive separation of components displaying dramatically different hydrophobicities, such as peptides, glycopeptides, and glycans.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid20456051,

title = {OsNOA1/RIF1 is a functional homolog of AtNOA1/RIF1: implication for a highly conserved plant cGTPase essential for chloroplast function},

author = {Hongjia Liu and Edward Lau and Maggie P Y Lam and Hung Chu and Sujuan Li and Guo Huang and Peng Guo and Junqi Wang and Liwen Jiang and Ivan K Chu and Clive Lo and Yuezhi Tao},

doi = {10.1111/j.1469-8137.2010.03264.x},

issn = {1469-8137},

year  = {2010},

date = {2010-07-01},

journal = {New Phytol},

volume = {187},

number = {1},

pages = {83--105},

abstract = {*The bacterial protein YqeH is a circularly permuted GTPase with homologs encoded by plant nuclear genomes. The rice homolog OsNOA1/RIF1 is encoded by the single-copy gene Os02g01440. OsNOA1/RIF1 is expressed in different tissues and is light-inducible. The OsNOA1/RIF1-EYFP fusion protein was targeted to chloroplasts in transgenic Arabidopsis plants. In addition, the rice homolog was able to rescue most of the growth phenotypes in an Arabidopsis rif1 mutant. *Rice (Oryza sativa) OsNOA1/RIF1 RNAi mutant seedlings were chlorotic with reduced pigment contents and lower photosystem II (PSII) efficiency. However, the expressions of the chloroplast-encoded genes rbcL, atpB, psaA and psbA were not affected. By contrast, reduced abundance of the chloroplast 16S rRNA was observed in the mutant. *Quantitative iTRAQ-LC-MS/MS proteomics investigations revealed proteome changes in the rice mutant consistent with the expected functional role of OsNOA1/RIF1 in chloroplast translation. The RNAi mutant showed significantly decreased expression levels of chloroplast-encoded proteins as well as nuclear-encoded components of chloroplast enzyme complexes. Conversely, upregulation of some classes of nonchloroplastic proteins, such as glycolytic and phenylpropanoid pathway enzymes, was detected. *Our work provides independent indications that a highly conserved nuclear-encoded cGTPase of likely prokaryotic origin is essential for proper chloroplast ribosome assembly and/or translation in plants.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}