A method for the comprehensive proteomic analysis of membrane proteins

Wu, Christine C.; MacCoss, Michael J.; Howell, Kathryn E.; Yates, John R.

doi:10.1038/nbt819

Cited by 655 publications

(583 citation statements)

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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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“…The brains of male Sprague-Dawley rats were dissected, and the hippocampal proteins were digested by proteinase K. 3 Five separate 12-cycle MudPITs on an Thermo Finnigan LCQ Deca were used to identify the peptides in this sample. The best analysis produced 86 846 spectra, yielding 1488 identifications above the high cutoffs.…”

Section: Sample Selectionmentioning

confidence: 99%

Influence of Basic Residue Content on Fragment Ion Peak Intensities in Low-Energy Collision-Induced Dissociation Spectra of Peptides

et al. 2004

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The primary utility of trypsin digestion in proteomics is that it cleaves proteins at predictable locations, but it is also notable for yielding peptides that terminate in basic arginine and lysine residues. Tryptic peptides fragment in ion trap tandem mass spectrometry to produce prominent Cterminal y series ions. Alternative proteolytic digests may produce peptides that do not follow these rules. In this study, we examine 2568 peptides generated through proteinase K digestion, a technique that produces a greater diversity of basic residue content in peptides. We show that the position of basic residues within peptides influences the peak intensities of b and y series ions; a basic residue near the N-terminus of a peptide can lead to prominent b series peaks rather than the intense y series peaks associated with tryptic peptides. The effects of presence and position for arginine, lysine, and histidine are explored separately and in combination. Arg shows the most dominant effects followed by His and then by Lys. Fragment ions containing basic residues produce more intense peaks than those without basic residues. Doubly charged precursor ions have generally been modeled as producing only singly charged fragment ions, but fragment ions that contain two basic residues may accept both protons during fragmentation. By characterizing the influence of basic residues on gasphase fragmentation of peptides, this research makes possible more accurate fragmentation models for peptide identification algorithms.Trypsin digestion is a standard step in many proteomic experiments. Proteins digested by this enzyme are predominantly cleaved C-terminal to arginine and lysine residues, yielding predictable collections of peptides.1 Tryptic peptides are particularly favored for tandem mass spectrometry because they yield spectra with prominent y series ions. Database search algorithms such as SEQUEST 2 have been tuned to work best with tryptic peptides. Alternative digestion techniques can be useful to analyze proteins that do not have many suitable cleavage sites for trypsin (such as membrane proteins) or to increase the diversity of peptides covering a protein region of interest. The cleavage specificities of other digestion enzymes may yield peptides that fragment differently than those produced by trypsin.One technique showing promise for proteomics is proteinase K digestion. 3 Proteinase K's specificity is more variable than trypsin, favoring amino acids with aromatic side chains as cleavage sites but targeting other residues as well. 4 The digestion time required is less than that of trypsin. Because of proteinase K's broader specificity, the diversity of peptides yielded by these digests is higher than in tryptic digests.The most common type of fragmentation used for proteomics is low-energy collision-induced dissociation (CID). In this technique, peptide ions of a particular mass-to-charge ratio are isolated and collided with noble gas atoms. Collisions add energy to the peptide ions and they fragment. The cleavage o...

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“…In addition to the interference encountered with mass spectrometric detection, concentrated urea or detergents denature many proteases and decrease their activity to cleave proteins. There are a number of reports describing technical advances to enable membrane protein analysis using the shotgun proteomics approach in which proteins are dissolved in a buffer containing surfactants or in an organic solvent, followed by protein digestion and liquid chromatography (LC) tandem mass spectrometry (MS/MS) analysis of the peptides [3][4][5][6][7][8][9][10][11][12]. Protein digestion was done with trypsin [3][4][5][6][7], cyanogen bromide (CNBr)-mediated enzymatic digestion [8 -11], and nonspecific proteinase K [12].…”

mentioning

confidence: 99%

Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification

Zhong

Marcus

2005

J. Am. Soc. Mass Spectrom.

152

142

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Simple and efficient digestion of proteins, particularly hydrophobic membrane proteins, is of significance for comprehensive proteome analysis using the bottom-up approach. We report a microwave-assisted acid hydrolysis (MAAH) method for rapid protein degradation for peptide mass mapping and tandem mass spectrometric analysis of peptides for protein identification. It uses 25% trifluoroacetic acid (TFA) aqueous solution to dissolve or suspend proteins, followed by microwave irradiation for 10 min. This detergent-free method generates peptide mixtures that can be directly analyzed by liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) without the need of extensive sample cleanup. LC-MALDI MS/MS analysis of the hydrolysate from 5 g of a model transmembrane protein, bacteriorhodopsin, resulted in almost complete sequence coverage by the peptides detected, including the identification of two posttranslational modification sites. Cleavage of peptide bonds inside all seven transmembrane domains took place, generating peptides of sizes amenable to MS/MS to determine possible sequence errors or modifications within these domains. Cleavage specificity, such as glycine residue cleavage, was observed. Terminal peptides were found to be present in relatively high abundance in the hydrolysate, particularly when low concentrations of proteins were used for MAAH. It was shown that these peptides could still be detected from MAAH of bacteriorhodopsin at a protein concentration of 1 ng/ l or 37 fmol/ l. To evaluate the general applicability of this method, it was applied to identify proteins from a membrane protein enriched fraction of cell lysates of human breast cancer cell line MCF7. With one-dimensional LC-MALDI MS/MS, a total of 119 proteins, including 41 membrane-associated or membrane proteins containing one to 12 transmembrane domains, were identified by MS/MS database searching based on matches of at least two peptides to a protein. (J Am Soc Mass Spectrom 2005, 16, 471-481)

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A method for the comprehensive proteomic analysis of membrane proteins

Cited by 655 publications

References 37 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

Influence of Basic Residue Content on Fragment Ion Peak Intensities in Low-Energy Collision-Induced Dissociation Spectra of Peptides

Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification

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