Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MS-based proteomics

What Can Proteomics Tell Us About Platelets?

“…21 Advantages are that, for peptides, (1) separation by chromatography, (2) ionization, and (3) fragmentation are more efficient, (4) their masses are less heterogeneously distributed, and (5) their identification through database searches is more straightforward. Complex peptide mixtures can be either directly analyzed by liquid chromatography-MS using long gradients 22 or fractionated by hyphenation of different separation techniques beforehand 23 to reduce sample complexity and increase depth and coverage of the proteome analysis.…”

“…However, especially when PTM-directed antibodies are used for the analysis of ubiquitination 33 or acetylation, 34 sensitivity and consequently large sample amounts can be an issue, even requiring several milligrams per sample for a comprehensive analysis. the generated data, it is important to monitor the entire workflow including (1) patient selection, (2) blood drawing, (3) platelet isolation and purity, (4) lysis and proteolytic digestion, 21 along with analytic steps such as (5) chemical labeling, 48 (6) prefractionation of the peptide mixture, (7) LC-MS analysis, 49 and (8) MS data interpretation. At present, costs and effort to conduct LC-MS-based proteome-wide analyses from individual patients are relatively high.…”

“…115 Owing to the enrichment provided by specific probes, activity-based protein profiling 116-121 B, These studies are mainly (40%) based on 2-dimensional gel electrophoresis (2-DE)/ difference gel electrophoresis (DIGE), 46,52,53,80,82,83,85,87,88,90,91,[93][94][95][96]98,102,106,110,112,113 whereas gelfree methods (23%) are still under-represented. 21,42,89,97,[114][115][116][117][118]121 Combination refers to studies using 2-DE/DIGE in combination with other approaches. COFRADIC indicates combined fractional diagonal chromatography; and MudPIT, multidimensional protein identification technology.…”

Exploring the potential of public proteomics data

“…One example is a study aimed at improving the understanding of the cleavage mechanism and performance of trypsin 37, 38, a crucial parameter in proteomic workflows. By inspecting the cleavage profile of all peptide identifications deposited in PRIDE, it was possible to train an algorithm that predicts trypsin cleavage sites, a functionality that is available through a web interface 39.…”

Discovery and elucidation of unknown protein modifications : brominated coumarin azide as a probe for alkynes

“…The standard workflow of MS peptide mapping includes 1) sample preparation; 2) digestion by a specific protease, such as trypsin; 3) HPLC separation of digested peptides followed by tandem MS/MS; 4) data analysis for identification and localization of the modification. 40 Over the past few years, great improvements in MS for proteomics analysis have been achieved regarding instrumentation and bioinformatics, [41][42] and some PTMs, such as phosphorylation, glycosylation, acetylation and ubiquitination can now be identified and localized in the protein sequence to a certain extent. [43][44] However, a significant amount of PTMs still remain difficult to analyze.…”