Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture

Dürr, Eberhard; Yu, Jun; Krasinska, Karolina; Carver, Lucy A.; Yates, John R.; Testa, Jacqueline E.; Oh, Phil; Schnitzer, Jan E.

doi:10.1038/nbt993

Cited by 401 publications

(397 citation statements)

References 31 publications

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“…In-depth studies of membrane proteins have proven to be difficult because of their low abundance and hydrophobicity 1,4 . However, recent advances in proteomic technologies make it possible to investigate proteins in this cell compartment, including previously unannotated membrane proteins, to an impressive depth [4][5][6][7][8][9] . In this study, we applied colloidal silica-bead coating 10 to enrich for conserved cell-surface-associated proteins in mouse neonatal and human fetal ventricular cardiomyocytes.…”

Section: Resultsmentioning

confidence: 99%

Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

et al. 2015

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Membrane proteins are crucial to heart function and development. Here we combine cationic silica-bead coating with shotgun proteomics to enrich for and identify plasma membrane-associated proteins from primary mouse neonatal and human fetal ventricular cardiomyocytes. We identify Tmem65 as a cardiac-enriched, intercalated disc protein that increases during development in both mouse and human hearts. Functional analysis of Tmem65 both in vitro using lentiviral shRNA-mediated knockdown in mouse cardiomyocytes and in vivo using morpholino-based knockdown in zebrafish show marked alterations in gap junction function and cardiac morphology. Molecular analyses suggest that Tmem65 interaction with connexin 43 (Cx43) is required for correct localization of Cx43 to the intercalated disc, since Tmem65 deletion results in marked internalization of Cx43, a shorter half-life through increased degradation, and loss of Cx43 function. Our data demonstrate that the membrane protein Tmem65 is an intercalated disc protein that interacts with and functionally regulates ventricular Cx43.

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Section: Resultsmentioning

confidence: 99%

Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

et al. 2015

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“…For example, it has been observed that two replicate MudPIT analyses will produce two sets of protein identifications with ,65% overlap [28,29]. Thirty-five percent of the proteins in the second analysis are likely to be novel compared to the first.…”

Section: Analytical Incompletenessmentioning

confidence: 99%

Guidelines for the next 10 years of proteomics

Wilkins¹,

Appel²,

Eyk³

et al. 2006

Proteomics

280

202

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In the last ten years, the field of proteomics has expanded at a rapid rate. A range of exciting new technology has been developed and enthusiastically applied to an enormous variety of biological questions. However, the degree of stringency required in proteomic data generation and analysis appears to have been underestimated. As a result, there are likely to be numerous published findings that are of questionable quality, requiring further confirmation and/or validation. This manuscript outlines a number of key issues in proteomic research, including those associated with experimental design, differential display and biomarker discovery, protein identification and analytical incompleteness. In an effort to set a standard that reflects current thinking on the necessary and desirable characteristics of publishable manuscripts in the field, a minimal set of guidelines for proteomics research is then described. These guidelines will serve as a set of criteria which editors of PROTEOMICS will use for assessment of future submissions to the Journal.

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“…By merging the two datasets along with the use of stricter SEQUEST filters (see Methods) for the PMT tags, the overall confidence of the resulting peptide/protein identifications (i.e., AMT tags) was improved. Combining multidimensional analyses has been shown elsewhere to increase the completeness of a proteomic analysis [32,37,38]. Recent analyses of human plasma and other samples provided the basis for estimating the false-positives rates for SEQUEST results, although the filter rules were similar, but not identical to those used here for populating the PMT tag database [39,40].…”

Section: Putative Mass and Time (Pmt) Tag Databasementioning

confidence: 95%

A proteomic study of the HUPO Plasma Proteome Project's pilot samples using an accurate mass and time tag strategy

et al. 2005

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Characterization of the human blood plasma proteome is critical to the discovery of routinely useful clinical biomarkers. We used an Accurate Mass and Time (AMT) tag strategy with high-resolution mass accuracy capillary liquid chromatography Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (cLC-FTICR MS) to perform a global proteomic analysis of pilot study samples as part of the HUPO Plasma Proteome Project. HUPO reference serum and citrated plasma samples from African Americans, Asian Americans, and Caucasian Americans were analyzed, in addition to a Pacific Northwest National Laboratory reference serum and plasma. The AMT tag strategy allowed us to leverage two previously published "shotgun" proteomics experiments to perform global analyses on these samples in triplicate in less than 4 days total analysis time. A total of 722 (22% with multiple peptide identifications) International Protein Index (IPI) redundant proteins, or 377 protein families by ProteinProphet, were identified over the 6 individual HUPO serum and plasma samples. The samples yielded a similar number of identified redundant proteins in the plasma samples (average 446 +/−23) as found in the serum samples (average 440+/−20). These proteins were identified by an average of 956+/−35 unique peptides in plasma and 930+/−11 unique peptides in serum. In addition to this high-throughput analysis, the AMT tag approach was used with a Z-score normalization to compare relative protein abundances. This analysis highlighted both known differences in serum and citrated plasma such as fibrinogens, and reproducible differences in peptide abundances from proteins such as soluble activin receptor-like kinase 7b and glycoprotein m6b. The AMT tag strategy not only improved our sample throughput, and provided a basis for estimated quantitation.

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Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture

Cited by 401 publications

References 31 publications

Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

Guidelines for the next 10 years of proteomics

A proteomic study of the HUPO Plasma Proteome Project's pilot samples using an accurate mass and time tag strategy

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