Taking the Shortcut for High-Throughput Shotgun Proteomic Analysis of Bacteria

Abstract

Currently, proteomic tools are able to establish a complete list of the most abundant proteins present in a sample, providing the opportunity to study at high resolution the physiology of any bacteria for which the genome sequence is available. For a comprehensive list, proteins should be first resolved into fractions that are then proteolyzed by trypsin. The resulting peptide mixtures are analyzed by a high-throughput tandem mass spectrometer that records thousands of MS/MS spectra for each fraction. These spectra are then assigned to peptides, which are used as evidence of the existence of proteins. In addition to generating a list of protein identifications, this shortcut to proteomics uses the number of spectra recorded for each protein to quantify the observations. Here, we describe one of the most simple sample preparation methods for high-throughput proteomics of bacteria, as well as the subsequent data processing to extract quantitative information based on the spectral count approach.

Functional distinctness in the exoproteomes of marine Synechococcus

“…Proteins in the remaining milieu were concentrated and purified by precipitation with trichloroacetic acid and run on SDS‐PAGE as previously described (Christie‐Oleza and Armengaud, 2010). Trypsin in‐gel proteolysis of the entire exoproteome was performed for the shotgun proteomics analysis as recommended (Hartmann et al ., 2014). NanoLC‐MS/MS experiments were performed using a LTQ‐Orbitrap XL hybrid mass spectrometer (ThermoFisher) coupled to an UltiMate 3000 LC system (Dionex‐LC Packings).…”

Proteogenomic insights into the core-proteome of female reproductive tissues from crustacean amphipods

“…The whole protein content from each well was extracted as a sole polyacrylamide band. The samples were destained, treated with iodoacetamide, and proteolyzed with Sequencing Grade Trypsin (Roche) using 0.01% ProteaseMAX surfactant (Promega) as described in [33]. The resulting peptide mixtures were diluted 1:20 in 0.1% trifluoroacetic acid.…”

“…Molecular ion peak lists were extracted with the Mascot Daemon software (version 2.4.0; Matrix Science) using the extract_msn.exe data import filter (Thermo). Data import filter options were set to 400 (minimum mass), 5000 (maximum mass), 0 (grouping tolerance), 0 (intermediate scans), and 1000 (threshold), as previously described [33]. Peptide assignation with MASCOT was done with the following parameters: full trypsin specificity, maximum of two missed cleavages, mass tolerances of 5 ppm on the parent ion and 0.5 Da on the MS/MS, static modification of carboxyamidomethylated cysteine(+57.0215), and oxidized methionine (+15.9949) as dynamic modification.…”

Deciphering the interactions between the Bacillus cereus linear plasmid, pBClin15, and its host by high-throughput comparative proteomics

“…3, three samples were taken at the time points corresponding to the early exponential growth phase (EE), late exponential growth phase (LE), and stationary phase (S). From the 18 resulting samples, all of the cellular soluble proteins and exoproteins obtained after trichloroacetic acid precipitation were processed separately by means of a standard gel-based procedure for trypsin proteolysis [43]. The 36 resulting peptide fractions corresponding to these proteomes were analyzed by high-throughput tandem mass spectrometry [25].…”