High Sensitivity Detection of Plasma Proteins by Multiple Reaction Monitoring of N-Glycosites

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 Nlabeled 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. Molecular & Cellular Proteomics 6:1809 -1817, 2007.The discovery and validation of protein biomarkers for the early detection of disease, for the assessment of a therapy's efficacy and side effects, and for drug development is one of the most active areas of proteomics research. Blood plasma represents a promising source of such markers as it has the advantage of being easily accessible. Plasma is circulating and exchanges analytes with cells and tissues within the entire body. In particular, proteins from dying cells and tumors that are shed, secreted, or otherwise released into the blood stream are expected to provide a "fingerprint" of the actual health status of the patient in general and of specific organs in particular. However, the large volume of fluid accounts for a substantial dilution of the analytes secreted or shed by an organ or a tissue. Low concentrations together with rapid clearance of some of the proteins create major challenges to a proteomics-based biomarker discovery approach that remain unresolved despite recent advances of proteomics technology. More specifically, the detection and quantification of proteins in plasma at concentrations in the ng/ml or sub-ng/ml range, at which meaningful biomarkers can realistically be expected (1), has remai...

Glycosylation as means of reducing sample complexity to enable quantitative proteomics

“…This was demonstrated by a recent study of biomarkers in human plasma, in which the N-glycosite capture method described above was used. It allowed detecting peptide in the low ng/ mL range, thus clearly indicating the importance of reduction of sample complexity in such analysis [5]. Similar experiments on plasma, without prior enrichment for a subproteome, showed a LOD in the 100 ng/mL range [13].…”

“…The high structural selectivity of the technique based on two levels of mass selection combined with an exquisite sensitivity allows the quantitative analysis of hundreds of peptides over a wide dynamic range of concentrations in one single LC-MS run [5]; with a linear response over near five orders of magnitude down to low ng/mL concentration was demonstrated in plasma samples.…”

Applications of targeted proteomics in systems biology and translational medicine

“…Upon purification, only the modified peptide is quantified. Stahl‐Zeng and coworkers applied minimal fractionation of isolated N‐glycosites to quantitate plasma proteins over five orders of magnitude, reaching sub‐ng/ml range 93. Zawadzka and coworkers used both targeted and discovery proteomics to quantify a set of approximately 60 phosphopeptides from healthy human plasma following offline chromatography and immobilized metal ion affinity chromatography for phosphopeptide enrichment 94.…”

A novel role for proteomics in the discovery of cell‐surface markers on stem cells: Scratching the surface

“…MS technology is capable of relative and absolute quantification of proteins [75]. Relative and absolute quantitative proteomic technologies (MRM [76], label-free [77], SILAC [78], iTRAQ [79], ICAT [80]) are key technologies, which should be included into iterative proteomic workflows that are designed to identify markers useful for the classification of stem cells. While a thorough discussion of quantitative proteomics is outside the scope of this review, readers are encouraged to refer to an excellent review on quantitative MS in proteomics that was recently published by Bantscheff et al [81].…”