Go to contents (site navigation)

 


Realised by ALMS™
developer of the AIDS-HIV Reference project
Abstract No.: FrOr-02
Speaker: Bruno Domon
Session: Biomarkers
Presentation date: Fri, Sep 1, 2006
Presentation time: 10:50 – 11:10

Novel Strategy for Rapid Screening and Quantification of Biomarkers in Plasma

Bruno Domon1, Jianru Zeng-Stahl2, Ruedi Aebersold1

1 ETH - Inst. Mol. Systems Biol., Zurich, Switzerland
2 Applied Biosystems, Darmstadt, Germany

Correspondence address: Bruno Domon, Institue for Molecular Systems Biology, ETH, HPT E73, Zurich, 8093 Switzerland.

Keywords: Biomarkers; Isotope Dilution; Mass Spectrometry, Ion Trap Quadrupole; Proteomic.

Novel aspect: MRM analyses on a QTrap instrument allowed detection and quantification of peptides in serum samples at 1050 attomol range.

 

In spite of the recent advances in technology, serum proteome analysis remains challenging because of the sample complexity and the wide range of protein concentrations. In our strategy, in-depth identification of peptides was performed on a high performance platform In a second step, as described in this study, putative biomarkers were quantified precisely in a larger number of samples to confirm the findings and to generate statistically significant data. The analyses were performed on a quadrupole-ion trap instrument in the multiple reaction monitoring mode. It allowed precise quantification of peptides in serum digests with high sensitivity and selectivity.

The glycopeptide capture method allowed selective isolation of a subset of peptides and thus reduced sample complexity to a level that enabled analysis in one single LC/MS(/MS) run. From the peptides previously identified, only a subset was selected for further evaluation, namely the proteotypic peptides (i.e. unambiguously associated with one single protein) and discriminating (i.e. show various level expression in a preliminary study).

Quantitative analyses of potential biomarkers were performed on a quadrupole ion trap instrument in the MRM mode. The method is high sensitivity as a result of removing chemical background in the first MS stage, and high selectivity through selection of pairs or precursor and y- fragment ions unique each peptide. Used in conjunction with isotopically labeled (13C) internal standards, the method showed a limit of quantification in the 10 to 50 attomol range. These values were obtained by spiking defined amount of isotopically labeled synthetic peptides in the serum N-linked glycopeptide fraction, and measuring the MRM signals of the standards and the corresponding endogenous peptides.

A relative large number of samples could be analyzed by this method in a straightforward manner to generate statistically significant results (biomarker specificity and sensitivity), thus validating or invalidating the individual markers, or helping designing panel of markers.

This detection limit was determined by monitoring up to one hundred transitions in one single LC/MS run, which corresponds to 25 peptides (i.e. two transitions per precursor for both internal standard and endogenous peptide). Broader screens were performed, by expanding the list of peptides to be monitored to several hundreds while compromising on the limit of detection. In addition, the system was programmed to acquire a full MS/MS spectrum when an ion of interest elutes to confirm the identity of the peptides.

This approach allows screening and evaluating biomarkers in plasma in high throughput mode with high selectivity and sensitivity.