title: Variable Digestion Strategies for Phosphoproteomics Analysis.
abstract: In recent years, mass spectrometry-based phosphoproteomics has propelled our knowledge about the regulation of cellular pathways. Nevertheless, typically applied bottom-up strategies have several limitations. Trypsin, the preferentially used proteolytic enzyme shows impaired cleavage efficiency in the vicinity of phosphorylation sites. Moreover, depending on the frequency and distribution of tryptic cleavage sites (Arg/Lys), generated peptides can be either too short or too long for confident identification using standard LC-MS approaches. To overcome these limitations, we introduce an alternative and simple approach based on the usage of the nonspecific serine protease subtilisin, which enables a fast and reproducible digestion and provides access to "hidden" areas of the proteome. Thus, in a single LC-MS experiment >1800 phosphopeptides were confidently identified and localized from 125 μg of HeLa digest, compared to >2100 sites after tryptic digestion. While the overlap was less than 20 %, subtilisin allowed the identification of many phosphorylation sites that are theoretically not accessible via tryptic digestion, thus considerably increasing the coverage of the phosphoproteome.
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund, Germany.|CAPES Foundation, Ministry of Education of Brazil, Caixa Postal 250, Brasília - DF, 70040-020, Brazil.
authors.forename: Humberto
authors.initials: H
authors.lastname: Gonczarowska-Jorge
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund, Germany.
authors.forename: Margherita
authors.initials: M
authors.lastname: Dell'Aica
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund, Germany.
authors.forename: Clarissa
authors.initials: C
authors.lastname: Dickhut
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund, Germany. rene.zahedi@isas.de.
authors.forename: René P
authors.initials: RP
authors.lastname: Zahedi
mesh_terms: D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D019295:Computational Biology; D030562:Databases, Protein; D006367:HeLa Cells; D006801:Humans; D010449:Peptide Mapping / Q000379:methods*; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D052616:Solid Phase Extraction; D020860:Subtilisin / Q000737:chemistry* / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014025:Titanium / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry; D057188:Workflow
chemical_list: D010748:Phosphopeptides; C009495:titanium dioxide; D014025:Titanium; D014357:Trypsin; D020860:Subtilisin
keywords: Phosphopeptide enrich ment; Phosphorylation; Subtilisin; Titanium dioxide; Trypsin