P23-M Limit of Quantitation for Low-Abundance Proteins in Plasma by Targeted MS

Biomarker discovery results in the creation of candidate lists of potential markers that must be subsequently verified in plasma.1 The most mature methods at present require abundant protein depletion and fractionation at the protein/peptide levels in order to detect and quantitate low ng/mL concentrations of plasma proteins by stable isotope dilution mass spectrometry. Sample-processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate bio-marker proteins were evaluated by adding five non-native proteins to immunoaffinity-depleted female plasma at varying concentrations (1000, 100, 50, 25, and 10 ng/mL). Each protein was monitored by one or more representative synthetic tryptic peptides labeled with [(13)C(6)]leucine or [(13)C(5)] valine. Following reduction, carbamidomethylation, and enzymatic digestion, two separate processing paths were compared. In path 1, digested plasma was diluted 1:10 and [(13)C] internal standards were added just prior to direct analysis by multiple reaction monitoring with LC-MS/MS (MRM LC-MS/MS). In path 2, peptides were separated by strong cation exchange, and [(13)C] internal standards were added to corresponding SCX fractions prior to analysis by MRM LC-MS/MS. Detection and quantitation by MRM used the response of at least two product ions from each of the signature peptides. Using processing path 1, we achieved detection and quantitation down to 50 ng/mL in depleted plasma. However, using processing path 2, we achieved detection and quantitation of all spiked proteins, including the non-native protein at 10 ng/mL. While analysis of non-fractionated plasma achieved higher recovery of those proteins detected in both processes, SCX fractionation at the peptide level clearly increases detection and LOQs for potential biomarker proteins in plasma.