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Low-bias phosphopeptide enrichment from scarce samples using plastic antibodies

Jing Chen1,*, Sudhirkumar Shinde2,*, Markus-Hermann Koch1, Martin Eisenacher1, Sara Galozzi1, Thilo Lerari1, Katalin Barkovits1, Prabal Subedi1, Rejko Krüger3,4Katja Kuhlmann1, Börje Sellergren2,*, Stefan Helling1,* & Katrin Marcus1,*

1Medizinisches Proteom-Center, Ruhr-University Bochum, Universitätsstr. 150, 44801 Bochum, Germany.
2Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, SE 205 06 Malmö, Sweden.
3Functional Neurogenomics Laboratory, Hertie-Institute for Clinical Brain Research, Eberhard Karls Universität Tuebingen and DZNE Tuebingen, Hoppe-Seyler-Str.3, 72076 Tuebingen, Germany. 4Clinical and Experimental Neuroscience, Luxembourg Centre for Systems Biomedicine, University of Luxembourg, University of Luxembourg, 7, avenue des Hauts-Fourneaux, L-4362 Esch-sur-Alzette.
*These authors contributed equally to this work. Correspondence and requests for materials should be addressed to B.S. (email: borje.sellergren@mah.se) or K.M. (email: This email address is being protected from spambots. You need JavaScript enabled to view it.)

Scientific Reports 5, Article number: 11438 (2015)
doi:10.1038/srep11438

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.






Antibody-Free Biomarker Determination: Exploring Molecularly Imprinted Polymers for Pro-Gastrin Releasing Peptide

Cecilia RossettiAbed Abdel QaderTrine Grønhaug HalvorsenBörje Sellergren§, and Léon Reubsaet*

 Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, NO-0316 Oslo, Norway
 Department of Environmental Chemistry and Analytical Chemistry, Institute for Environmental Research (INFU), Technical University of Dortmund, D-44221 Dortmund, Germany
§ Department of Biomedical Sciences, Faculty of Health and Society, University of Malmö, 205 06 Malmö,Sweden


Anal. Chem., 2014, 86 (24), pp 12291–12298
DOI: 10.1021/ac503559c

Abstract

Biomarker mass spectrometry assays are in high demand, and analysis of pro-gastrin releasing peptide (ProGRP) as a small cell lung cancer marker has been recently investigated by mass spectrometry after immunoextraction. In this article, we introduce an assay based on molecularly imprinted polymers (MIPs) targeting the proteotypic peptide of ProGRP as a possible alternative to current immuno-based assay. The MIPs were prepared by surface-initiated reversible addition–fragmentation chain transfer polymerization and were introduced as sorbents for the cleanup and enrichment of a ProGRP signature peptide from tryptically treated serum samples. The use of an appropriate solid-phase extraction protocol allowed specific extraction of the target peptide while depleting other peptides that arose from the sample digestion, hence resulting in reduced background. The selective extraction of a ProGRP signature peptide, after digestion of serum samples, translates into a time- and cost-effective method suited for bottom-up analysis wherever targeted peptide extraction from complex matrices is required.

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