A comparative study of phosphopeptide-selective techniques for a sub-proteome of a complex biological sample.
2016 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 408, no 9, 2347-2356 p.Article in journal (Refereed) Published
Phosphorylation of proteins is important for controlling cellular signaling and cell cycle regulatory events. The process is reversible and phosphoproteins normally constitute a minor part of the global proteome in a cell. Thus, sample preparation techniques tailored for phosphoproteome studies are continuously invented and evaluated. This paper aims at evaluating the performances of the most popular techniques for phospho-enrichments in sub-proteome analysis, such as viral proteomes expressed in human cells during infection. A two-species sample of Adenovirus type 2 infected human cells was used, and in-solution digestion, strong cation exchange (SCX), and electrostatic repulsion hydrophilic interaction chromatography (ERLIC) fractionation, and subsequent enrichment by TiO2, were compared with SDS-PAGE fractionation and in-gel digestion. Evaluation was focused on phosphopeptide detection in the sub-proteome. The results showed that the SCX+TiO2 or ERLIC+TiO2 combinations had the highest enrichment efficiencies, but SDS-PAGE fractionation and in-gel digestion resulted in the highest number of identified proteins and phosphopeptides. Furthermore, the study demonstrates the usefulness of applying as many orthogonal techniques as possible in deep phosphoproteome analysis, since the overlap between approaches was low.
Place, publisher, year, edition, pages
2016. Vol. 408, no 9, 2347-2356 p.
Research subject Chemistry with specialization in Analytical Chemistry
IdentifiersURN: urn:nbn:se:uu:diva-281316DOI: 10.1007/s00216-016-9333-2ISI: 000372598600017PubMedID: 26886742OAI: oai:DiVA.org:uu-281316DiVA: diva2:913792
FunderMagnus Bergvall FoundationSwedish Research Council, 621-2011-4423