Identification and relative quantification of phosphopeptides by a combination of multi‐protease digestion and isobaric labeling

Abstract

A workflow for the straightforward comparative analysis of protein phosphorylation in samples of low complexity, e.g. isolated proteins, was developed. The workflow is transferable to other posttranslational modifications.

Responses in growth performance and nutrient digestibility to a multi-protease supplementation in amino acid-deficient broiler diets

“…The utilization of multi-proteases has been proven by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (via the separation of phosphoproteins) to involve trypsin alone, improving the protein sequence coverage due to the increasing diversity of the peptide sequences, and this can facilitate the identification of protein sequences lacking tryptic cleavage sites by application of different cleavage site specificities [40,41]. Non-starch polysaccharides (NSP) in cereals and soybeans provide physical barriers to prevent the action of exogenous enzymes on starch and protein [42].…”

Multi-protease Approach for the Improved Identification and Molecular Characterization of Small Proteins and Short Open Reading Frame-Encoded Peptides

“…Bartel et al used alternative proteases to trypsin, which reduced the spectral count for larger proteins and led to an improved identification of small proteins . Moreover, the application of multiple proteases has been established for several specific issues, e.g., phosphoproteomics, cross-linking mass spectrometry, C-terminomics, or paleoproteomics . Several tools for the in silico prediction of a suitable protease have been introduced. , However, due to the factors such as ionization efficiency or chromatographic co-elution, in silico -predicted peptides are not necessarily also identifiable in a real-life wet-lab experiment.…”

“…24 Bartel et al used alternative proteases to trypsin, which reduced the spectral count for larger proteins and led to an improved identification of small proteins. 13 Moreover, the application of multiple proteases has been established for several specific issues, e.g., phosphoproteomics, 25 cross-linking mass spectrometry, 26 C-terminomics, 27 or paleoproteomics. 28 Several tools for the in silico prediction of a suitable protease have been introduced.…”

Recent advances in phosphoproteomics and application to neurological diseases

“…25 Several groups have demonstrated enhanced proteomic identification and/or PTM localization rates when using alternative proteases such as Lys-C, Asp-N, Glu-C, chymotrypsin, elastase, and subtilisin, often in combination with trypsin. 24, 26–29 Aside from choice of protease, digestion protocols can be optimized according to temperature, denaturing reagent, time, and so forth. Filtered assisted sample preparation (FASP) and phase transfer surfactant-aided extraction (PTS) represent significant advances for phosphoproteome analysis of membrane proteins and tissue, whether fresh 30 or formalin-fixed/paraffin-embedded.…”