Halobacterium salinarum NRC-1 PeptideAtlas: Toward Strategies for Targeted Proteomics and Improved Proteome Coverage

Abstract

The relatively small numbers of proteins and fewer possible posttranslational modifications in microbes provides a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a Peptide Atlas (PA) for 62.7% of the predicted proteome of the extremely halophilic archaeon Halobacterium salinarum NRC-1 by compiling approximately 636,000 tandem mass spectra from 497 mass spectrometry runs in 88 experiments. Analysis of the PA with respect to biophysical properties of constituent peptides, functional properties of parent proteins of detected peptides, and performance of different mass spectrometry approaches has helped highlight plausible strategies for improving proteome coverage and selecting signature peptides for targeted proteomics. Notably, discovery of a significant correlation between absolute abundances of mRNAs and proteins has helped identify low abundance of proteins as the major limitation in peptide detection. Furthermore we have discovered that iTRAQ labeling for quantitative proteomic analysis introduces a significant bias in peptide detection by mass spectrometry. Therefore, despite identifying at least one proteotypic peptide for almost all proteins in the PA, a context-dependent selection of proteotypic peptides appears to be the most effective approach for targeted proteomics.

Quantification of human serum transferrin using liquid chromatography–tandem mass spectrometry based targeted proteomics

“…However, it should be noted that this peptide sequence is also observed in other primates. Finally, this selected peptide was also consistent with the previous evidence (http://www.peptideatlas.org) [21].…”

Internal RNAs overlapping coding sequences can drive the production of alternative proteins in archaea

“…Instead of relying on concordance between predicted and measured MW of translation products, we searched H. salinarum MS datasets for tryptic and semi-tryptic peptides since one would expect intraORF N-termini to generate different peptide sizes to the overlapping region of the full-length ORF due to the lack of a protease site at the N-terminus. We used two large-scale sources: PeptideAtlas database (www.peptideatlas.org [38]), which accumulated diverse H. salinarum NRC-1 proteomics data; and a more recent SWATH ( S equential W indow A cquisition of all Th eoretical Fragment Ion Spectra) high-depth data (PRIDE database accession PXD003667) [39] for H. salinarum R1.…”

“…Semi-tryptic peptides, i.e . sequences with arginine (R) or lysine (K) as their last C-terminal amino acid residue but not at N-terminal, were searched in PeptideAtlas curated database (www.peptideatlas.org) against the latest public H. salinarum NRC-1 build (2014–11) [38] and against H. salinarum R1 SWATH data (PRIDE ID PXD003667) [39]. Two semi-tryptic peptides variations were considered, full peptides and methionine cleaved peptides (thus missing first M residue).…”

Genotyping Common and Rare Variation Using Overlapping Pool Sequencing

“…We and others have found that precision is generally a function of mean abundance in iTRAQ data [11,[36][37][38][39][40]. This varying precision is not evident in standard residual plots, but is evident in per-MS experiment plots.…”