To elute or not to elute in immunocapture bottom-up LC-MS.

Autor: Levernæs MCS; Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, PO Box 1068 Blindern, 0316 Oslo, Norway. Electronic address: m.c.s.levernas@farmasi.uio.no., Broughton MN; Department of Medical Biochemistry, Norwegian Radium Hospital, Oslo University Hospital, PO Box 4953 Nydalen, 0424 Oslo, Norway. Electronic address: nordlund.marianne@gmail.com., Reubsaet L; Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, PO Box 1068 Blindern, 0316 Oslo, Norway. Electronic address: leon.reubsaet@farmasi.uio.no., Halvorsen TG; Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, PO Box 1068 Blindern, 0316 Oslo, Norway. Electronic address: t.g.halvorsen@farmasi.uio.no.
Jazyk: angličtina
Zdroj: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences [J Chromatogr B Analyt Technol Biomed Life Sci] 2017 Jun 15; Vol. 1055-1056, pp. 51-60. Date of Electronic Publication: 2017 Apr 14.
DOI: 10.1016/j.jchromb.2017.03.044
Abstrakt: Immunocapture-based bottom-up LC-MS is a promising technique for the quantification of low abundant proteins. Magnetic immunocapture beads provide efficient enrichment from complex samples through the highly specific interaction between the target protein and its antibody. In this article, we have performed the first thorough comparison between digestion of proteins while bound to antibody coated beads versus after elution from the beads. Two previously validated immunocapture based MS methods for the quantification of pro-gastrin releasing peptide (ProGRP) and human chorionic gonadotropin (hCG) were used as model systems. The tryptic peptide generation was shown to be protein dependent and influenced by protein folding and accessibility towards trypsin both on-beads and in the eluate. The elution of proteins bound to the beads was also shown to be incomplete. In addition, the on-beads digestion suffered from non-specific binding of the trypsin generated peptides. A combination of on-beads digestion and elution may be applied to improve both the quantitative (peak area of the signature peptides) and qualitative yield (number of missed cleavages, total number of identified peptides, coverage, signal intensity and number of zero missed cleavage peptides) of the target proteins. The quantitative yield of signature peptides was shown to be reproducible in all procedures tested.
(Copyright © 2017 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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