On-line immunoaffinity-liquid chromatography-mass spectrometry for identification of amyloid disease markers in biological fluids

Jette W. Sen, H. Robert Bergen, Niels H.H. Heegaard

Research output: Contribution to journalArticle

27 Scopus citations

Abstract

Disease-specific alterations in proteins and peptides such as the appearance of new isoforms, changed relative concentrations of known isoforms, or changed catabolism characterize the group of protein precipitation disorders collectively known as amyloidoses. The goal of this study was to develop an approach for isolating and characterizing the pool of isoforms of a polypeptide of interest from biological fluids for use in development of diagnostic markers and elucidation of pathogenesis. For this purpose, we employed an on-line immunoaffinity-liquid chromatography-mass spectrometry (IA-LC-MS) modular approach using antibodies binding populations of protein isoforms. In this system, crude biological samples, e.g., serum, may be injected and subjected to fast hands-off analysis. The setup consists of an optional preclear column for removal of unspecific binding components, an immunoaffinity column, a short cartridgelike reversed-phase column, and an electrospray time-of-flight mass spectrometer. We have tested the system for the automated analysis of three amyloid-related polypeptides, serum amyloid P component, amyloid β-peptide, and β2-microglobulin, and we show the feasibility of detection of altered isoforms or determination of relative abundance of isoforms of the proteins from serum or cerebrospinal fluid samples. For each new protein investigated, the only change needed in the system is a new antibody or antibody mixture and the selection of a reversed-phase cartridge of appropriate hydrophobicity.

Original languageEnglish (US)
Pages (from-to)1196-1202
Number of pages7
JournalAnalytical Chemistry
Volume75
Issue number5
DOIs
StatePublished - Mar 1 2003

ASJC Scopus subject areas

  • Analytical Chemistry

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