title: Low-bias phosphopeptide enrichment from scarce samples using plastic antibodies.
abstract: Phosphospecific enrichment techniques and mass spectrometry (MS) are essential tools for comprehending the cellular phosphoproteome. Here, we report a fast and simple approach for low sequence-bias phosphoserine (pS) peptide capture and enrichment that is compatible with low biological or clinical sample input. The approach exploits molecularly imprinted polymers (MIPs, "plastic antibodies") featuring tight neutral binding sites for pS or pY that are capable of cross-reacting with phosphopeptides of protein proteolytic digests. The versatility of the resulting method was demonstrated with small samples of whole-cell lysate from human embryonic kidney (HEK) 293T cells, human neuroblastoma SH-SY5Y cells, mouse brain or human cerebrospinal fluid (CSF). Following pre-fractionation of trypsinized proteins by strong cation exchange (SCX) chromatography, pS-MIP enrichment led to the identification of 924 phosphopeptides in the HEK 293T whole-cell lysate, exceeding the number identified by TiO2-based enrichment (230). Moreover, the phosphopeptides were extracted with low sequence bias and showed no evidence for the characteristic preference of TiO2 for acidic amino acids (aspartic and glutamic acid). Applying the method to human CSF led to the discovery of 47 phosphopeptides belonging to 24 proteins and revealed three previously unknown phosphorylation sites.
authors.affiliation: Medizinisches Proteom-Center, Ruhr-University Bochum, Universitätsstr. 150, 44801 Bochum, Germany.
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, SE 205 06 Malmö, Sweden.
authors.forename: Sudhirkumar
authors.initials: S
authors.lastname: Shinde
authors.affiliation: Medizinisches Proteom-Center, Ruhr-University Bochum, Universitätsstr. 150, 44801 Bochum, Germany.
authors.forename: Markus-Hermann
authors.initials: MH
authors.lastname: Koch
authors.affiliation: Medizinisches Proteom-Center, Ruhr-University Bochum, Universitätsstr. 150, 44801 Bochum, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Eisenacher
authors.affiliation: Medizinisches Proteom-Center, Ruhr-University Bochum, Universitätsstr. 150, 44801 Bochum, Germany.
authors.forename: Sara
authors.initials: S
authors.lastname: Galozzi
authors.affiliation: Medizinisches Proteom-Center, Ruhr-University Bochum, Universitätsstr. 150, 44801 Bochum, Germany.
authors.forename: Thilo
authors.initials: T
authors.lastname: Lerari
authors.affiliation: Medizinisches Proteom-Center, Ruhr-University Bochum, Universitätsstr. 150, 44801 Bochum, Germany.
authors.forename: Katalin
authors.initials: K
authors.lastname: Barkovits
authors.affiliation: Medizinisches Proteom-Center, Ruhr-University Bochum, Universitätsstr. 150, 44801 Bochum, Germany.
authors.forename: Prabal
authors.initials: P
authors.lastname: Subedi
authors.affiliation: Functional Neurogenomics Laboratory, Hertie-Institute for Clinical Brain Research, Eberhard Karls Universität Tuebingen and DZNE Tuebingen, Hoppe-Seyler-Str.3, 72076 Tuebingen, Germany.|Clinical and Experimental Neuroscience, Luxembourg Centre for Systems Biomedicine, University of Luxembourg, University of Luxembourg, 7, avenue des Hauts-Fourneaux, L-4362 Esch-sur-Alzette.
authors.forename: Rejko
authors.initials: R
authors.lastname: Krüger
authors.affiliation: Medizinisches Proteom-Center, Ruhr-University Bochum, Universitätsstr. 150, 44801 Bochum, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Kuhlmann
authors.affiliation: Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, SE 205 06 Malmö, Sweden.
authors.forename: Börje
authors.initials: B
authors.lastname: Sellergren
authors.affiliation: Medizinisches Proteom-Center, Ruhr-University Bochum, Universitätsstr. 150, 44801 Bochum, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Helling
authors.affiliation: Medizinisches Proteom-Center, Ruhr-University Bochum, Universitätsstr. 150, 44801 Bochum, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Marcus
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D002555:Cerebrospinal Fluid / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D054802:Molecular Imprinting / Q000379:methods*; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010969:Plastics / Q000737:chemistry*; D040901:Proteomics; D052616:Solid Phase Extraction; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000906:Antibodies; D010748:Phosphopeptides; D010969:Plastics; D010768:Phosphoserine