Analysis of in vivo-derived amyloid-β polypeptides by on-line two-dimensional chromatography-mass spectrometry

Nigel J. Clarke, Frank W. Crow, Steven Younkin, Stephen Naylor

Research output: Contribution to journalArticlepeer-review

22 Scopus citations


The presence of senile plaques composed of amyloid-β (Aβ) polypeptides within brain tissue is normally used as a definitive postmortem diagnosis for Alzheimer's Disease (AD). Therefore, these polypeptides have been investigated as potential biomarkers of the disease state. However, at present, there is a lack of a robust assay for the detection of such polypeptides derived from in vivo sources. Such an assay is essential for analysis of biological samples from model AD systems. To overcome this problem we have developed a new single-step assay utilizing two dimensional-chromatography in conjunction with mass spectrometry. The method consists of on-line size-exclusion chromatography (SEC) to provide initial separation of analytes from the sample (based on their molecular weight) coupled with sample preconcentration prior to analysis by microbore high-performance liquid chromatography-mass spectrometry (HPLC-MS). This provides an extremely versatile and powerful assay which can separate specific analytes from cell lysate in a single step without further sample handling. The use of mass spectrometry as the detection system yields much more structural information than can be obtained from traditional ELISA and sandwich ELISA antibody assays. Furthermore, the on-line sample cleanup protocol minimizes sample handling and facilitates assay automation. Utilizing this new assay we have been able to detect Aβ 1-40 and Aβ 1-42 at cellular concentration levels directly from cell lysates. Moreover, we have detected multiple peptide responses within the same analysis, some of which have been tentatively identified as other ragged C-termini Aβ polypeptides derived from Aβ 1-42, based on their molecular weight, as well as oxidized Aβ polypeptides.

Original languageEnglish (US)
Pages (from-to)32-39
Number of pages8
JournalAnalytical Biochemistry
Issue number1
StatePublished - Nov 1 2001

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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