Abstract
The detection and quantification of plasma (serum) proteins at or below the ng/ml concentration range are of critical importance for the discovery and evaluation of new protein biomarkers. This has been achieved either by the development of high sensitivity ELISA or other immunoassays for specific proteins or by the extensive fractionation of the plasma proteome followed by the mass spectrometric analysis of the resulting fractions. The first approach is limited by the high cost and time investment for assay development and the requirement of a validated target. The second, although reasonably comprehensive and unbiased, is limited by sample throughput. Here we describe a method for the detection of plasma proteins at concentrations in the ng/ml or sub-ng/ml range and their accurate quantification over 5 orders of magnitude. The method is based on the selective isolation of N-glycosites from the plasma proteome and the detection and quantification of targeted peptides in a quadrupole linear ion trap instrument operated in the multiple reaction monitoring (MRM) mode. The unprecedented sensitivity of the mass spectrometric analysis of minimally fractionated plasma samples is the result of the significantly reduced sample complexity of the isolated N-glycosites compared with whole plasma proteome digests and the selectivity of the MRM process. Precise quantification was achieved via stable isotope dilution by adding (13)C- and/or (15)N-labeled reference analytes. We also demonstrate the possibility of significantly expanding the number of MRM measurements during one single LC-MS run without compromising sensitivity by including elution time constraints for the targeted transitions, thus allowing quantification of large sets of peptides in a single analysis.
Highlights
The detection and quantification of plasma proteins at or below the ng/ml concentration range are of critical importance for the discovery and evaluation of new protein biomarkers
As an alternative to chromatographic or electrophoretic fractionation of the plasma proteome we recently introduced the selective isolation of the deglycosylated forms of those peptides that are N-glycosylated in intact proteins (N-glycosites) by a solid-phase method and their subsequent mass spectrometric analysis [10]
Assessment of Sample Quality—The objective of the study was to identify and quantify selected plasma proteins at high sensitivity by applying the multiple reaction monitoring (MRM) technique to populations of N-glycosite peptides isolated from the plasma proteome
Summary
The detection and quantification of plasma (serum) proteins at or below the ng/ml concentration range are of critical importance for the discovery and evaluation of new protein biomarkers This has been achieved either by the development of high sensitivity ELISA or other immunoassays for specific proteins or by the extensive fractionation of the plasma proteome followed by the mass spectrometric analysis of the resulting fractions. This approach has the advantage that the assay, once developed, can be applied to high numbers of samples with a high degree of robustness and at low cost Such assays are sensitive with limits of detection in the sub-ng/ml range being routinely achieved. This approach is conceptually simple, its successful practical im-
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