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Development of an enrichment method for endogenous phosphopeptide characterization in human serum

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Abstract

The work describes the development of an enrichment method for the analysis of endogenous phosphopeptides in serum. Endogenous peptides can play significant biological roles, and some of them could be exploited as future biomarkers. In this context, blood is one of the most useful biofluids for screening, but a systematic investigation of the endogenous peptides, especially phosphorylated ones, is still lacking, mainly due to the lack of suitable analytical methods. Thus, in this paper, different phosphopeptide enrichment strategies were pursued, based either on metal oxide affinity chromatography (MOAC, in the form of commercial TiO2 spin columns or magnetic graphitized carbon black-TiO2 composite), or on immobilized metal ion affinity chromatography (IMAC, in the form of Ti4+-IMAC magnetic material or commercial Fe3+-IMAC spin columns). While MOAC strategies proved completely unsuccessful, probably due to interfering phospholipids displacing phosphopeptides, the IMAC materials performed very well. Different sample preparation strategies were tested, comprising direct dilution with the loading buffer, organic solvent precipitation, and lipid removal from the matrix, as well as the addition of phosphatase inhibitors during sample handling for maximized endogenous phosphopeptide enrichment. All data were acquired by a shotgun peptidomics approach, in which peptide samples were separated by reversed-phase nanoHPLC hyphenated with high-resolution tandem mass spectrometry. The devised method allowed the identification of 176 endogenous phosphopeptides in fresh serum added with inhibitors by the direct dilution protocol and the Ti4+-IMAC magnetic material enrichment, but good results could also be obtained from the commercial Fe3+-IMAC spin column adapted to the batch enrichment protocol.

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The authors declare that they have no conflicts of interest.

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This work was supported by PRIN 2015, project number 2015TWP83Z.

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Correspondence to Susy Piovesana.

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No human patients or animals were involved in this study. Blood samples were provided after informed consent was obtained according to the Ethics Committee of the Umberto I Hospital, (Sapienza University of Rome).

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Published in the topical collection celebrating ABCs 16th Anniversary.

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La Barbera, G., Capriotti, A.L., Cavaliere, C. et al. Development of an enrichment method for endogenous phosphopeptide characterization in human serum. Anal Bioanal Chem 410, 1177–1185 (2018). https://doi.org/10.1007/s00216-017-0822-8

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