title: A comparative study of phosphopeptide-selective techniques for a sub-proteome of a complex biological sample.
abstract: 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.
authors.affiliation: Department of Chemistry-BMC, Analytical Chemistry and Science for Life Laboratory, Uppsala University, Box 599, 75124, Uppsala, Sweden.
authors.forename: Malin
authors.initials: M
authors.lastname: Källsten
authors.affiliation: Department of Chemistry-BMC, Analytical Chemistry and Science for Life Laboratory, Uppsala University, Box 599, 75124, Uppsala, Sweden.
authors.forename: Jonas
authors.initials: J
authors.lastname: Bergquist
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Rudbeck Laboratory, 751 85, Uppsala, Sweden.
authors.forename: Hongxing
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Department of Chemistry-BMC, Analytical Chemistry and Science for Life Laboratory, Uppsala University, Box 599, 75124, Uppsala, Sweden.
authors.forename: Anne
authors.initials: A
authors.lastname: Konzer
authors.affiliation: Department of Chemistry-BMC, Analytical Chemistry and Science for Life Laboratory, Uppsala University, Box 599, 75124, Uppsala, Sweden. sara.lind@kemi.uu.se.
authors.forename: Sara Bergström
authors.initials: SB
authors.lastname: Lind
mesh_terms: D002460:Cell Line; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D010748:Phosphopeptides / Q000737:chemistry*; D020543:Proteome; D053719:Tandem Mass Spectrometry
chemical_list: D010748:Phosphopeptides; D020543:Proteome
keywords: ERLIC; High-resolution mass spectrometry; Phosphoproteomics; SCX; SDS-PAGE; Sub-proteome analysis; TiO2