TY - JOUR
T1 - Development of an enrichment method for endogenous phosphopeptide characterization in human serum
AU - La Barbera, Giorgia
AU - Capriotti, Anna Laura
AU - Cavaliere, Chiara
AU - Ferraris, Francesca
AU - Laus, Michele
AU - Piovesana, Susy
AU - Sparnacci, Katia
AU - Laganà, Aldo
PY - 2018/1/1
Y1 - 2018/1/1
N2 - 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.
AB - 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.
KW - Endogenous serum phosphopeptides
KW - Magnetic solid-phase extraction
KW - NanoHPLC-MS/MS
KW - Phosphopeptide enrichment
KW - Phosphoproteomics
KW - Ti-IMAC
UR - http://www.scopus.com/inward/record.url?scp=85040228507&partnerID=8YFLogxK
U2 - 10.1007/s00216-017-0822-8
DO - 10.1007/s00216-017-0822-8
M3 - Journal article
C2 - 29318361
AN - SCOPUS:85040228507
SN - 1618-2642
VL - 410
SP - 1177
EP - 1185
JO - Analytical and Bioanalytical Chemistry
JF - Analytical and Bioanalytical Chemistry
IS - 3
ER -