Article (Scientific journals)
Low-bias phosphopeptide enrichment from scarce samples using plastic antibodies
Chen, Jing; Shinde, Sudhirkumar; Koch, Markus-Hermann et al.
2015In Scientific Reports, 5, p. 11438
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Abstract :
[en] 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.
Research center :
- Luxembourg Centre for Systems Biomedicine (LCSB): Clinical & Experimental Neuroscience (Krüger Group)
Disciplines :
Genetics & genetic processes
Author, co-author :
Chen, Jing
Shinde, Sudhirkumar
Koch, Markus-Hermann
Eisenacher, Martin
Galozzi, Sara
Lerari, Thilo
Barkovits, Katalin
Subedi, Prabal
KRÜGER, Rejko ;  University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit
Kuhlmann, Katja
Sellergren, Borje
Helling, Stefan
Marcus, Katrin
More authors (3 more) Less
External co-authors :
yes
Language :
English
Title :
Low-bias phosphopeptide enrichment from scarce samples using plastic antibodies
Publication date :
2015
Journal title :
Scientific Reports
ISSN :
2045-2322
Publisher :
Nature Publishing Group, London, United Kingdom
Volume :
5
Pages :
11438
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBilu :
since 17 July 2015

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