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Isoform differentiation of intact AGP from human serum by capillary electrophoresis–mass spectrometry

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Abstract

Human AGP is an acidic glycoprotein mainly produced by liver that presents a high degree of heterogeneity. It can present different amino acid sequences and has five N-glycosylation sites leading to a wide range of different protein isoforms. AGP structure and composition has been widely studied due to its drug-binding behavior and relation with disease. However, so far, the characterization has been performed only on protein fragments, i.e., the peptide or glycan level. Here, the analysis of intact human AGP purified from human serum is performed by capillary electrophoresis–time-of-flight mass spectrometry. In this way, it is possible to characterize more than 150 human AGP isoforms, differing both in the amino acid sequence and in the glycosylation. The detected masses could be attributed unequivocally to an overall composition based on the combination of the analysis of the released glycans and the characterization of the deglycosylated protein. Different AGP samples purified from human serum were characterized and compared. High inter-individual variability among AGP isoforms expression was observed. The presented method enables for the first time clinical studies based on detailed isoform distribution of intact glycoproteins.

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Abbreviations

AGP:

Alpha-1-acid glycoprotein

SA:

Sialic acid

CE:

Capillary electrophoresis

MS:

Mass spectrometry

CZE:

Capillary zone electrophoresis

MALDI-MS:

Matrix-assisted laser desorption mass spectrometry

ESI-MS:

Electrospray mass spectrometry

CZE-ESI-MS:

Capillary zone electrophoresis–electrospray mass spectrometry

PGNase F:

Peptide N-glycosidase F

BGE:

Background electrolytes

BPE:

Base peak electropherogram

EIE:

Extracted ion electropherogram

Ant:

antennae

HexHexNAc:

hexose-N-acetylhexosamine

DMA-EPyM:

acrylamide–pyrrolidine methacrylate

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Acknowledgments

Sara Ongay acknowledges CSIC for a grant for a short stay in the University of Aalen, Germany. We thank Carolin Huhn (University of Aalen, now Forschungszentrum Jülich, Germany) for support, Alejandro Cifuentes (IFI, CSIC, Madrid, Spain) for donation of the copolymer coating and M. Val Toledo (Hospital Ramón y Cajal, Madrid, Spain) and Enrique Caso (Complejo Hospitalario Universitario, Vigo, Spain) for donation of the serum samples. We thank Ute Pyell, University of Marburg for the loan of CE instrumentation. Financial support from the Spanish Ministry of Science and Innovation (projects CTQ 2006-05214, HH 2006-0013, and PSE-010000-2008-6) and Comunidad de Madrid (project S-GEN-0247-2006) is acknowledged.

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Authors and Affiliations

  1. Institute of Organic Chemistry (C.S.I.C.), Juan de la Cierva 3, 28006, Madrid, Spain

    Sara Ongay

  2. University of Aalen, Beethovenstraβe 1, 73430, Aalen, Germany

    Christian Neusüβ

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  1. Sara Ongay
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  2. Christian Neusüβ
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Correspondence to Christian Neusüβ.

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Ongay, S., Neusüβ, C. Isoform differentiation of intact AGP from human serum by capillary electrophoresis–mass spectrometry. Anal Bioanal Chem 398, 845–855 (2010). https://doi.org/10.1007/s00216-010-3948-5

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  • DOI: https://doi.org/10.1007/s00216-010-3948-5

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