"Product ion monitoring" assay for prostate-specific antigen in serum using a linear ion-trap.

Abstract:

:While numerous strategies exist for biomarker discovery, the bottleneck to product development and routine use at the clinic is in the verification phase of candidate biomarkers. The aim of this study was to establish a robust and high-throughput product ion monitoring (PIM) assay that is potentially capable of rapidly verifying candidates from discovery phase experiments. Using prostate-specific antigen (PSA), a model biomarker, and a routinely used mass spectrometer for discovery platforms, an ion trap (LTQ, Thermo), the utility of this instrument to perform PIM was explored. The proteotypic doubly charged intact peptide LSEPAELTDAVK ( m/ z 637) fragmenting to m/ z 943 (PAELTDAVK) was monitored. A limit of detection of 10 attomoles with a coefficient of variation (CV) of <20% was obtained for a purified recombinant PSA digest. Immunoextraction of endogenous PSA from serum using a monoclonal antibody on a 96-well microtiter plate, followed by PIM on the LTQ, enabled quantification of PSA down to less than 1 ng/mL with a limit of detection of 0.1 ng/mL and CVs < 20%. Mascot searching and ion ratio confirmation further supported the conclusion that the quantified moiety in serum was the PSA peptide. We conclude that this methodology could be adapted quickly and easily to other candidates, thus providing a much needed technology to bridge the gap between discovery and validation platforms.

journal_name

J Proteome Res

authors

Kulasingam V,Smith CR,Batruch I,Buckler A,Jeffery DA,Diamandis EP

doi

10.1021/pr7005999

subject

Has Abstract

pub_date

2008-02-01 00:00:00

pages

640-7

issue

2

eissn

1535-3893

issn

1535-3907

journal_volume

7

pub_type

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