Mass spectrometry (MS)-based protein quantitation is increasingly being employed to verify candidate protein biomarkers. Multiple or selected reaction monitoring-mass spectrometry (MRM-MS or SRM-MS) with isotopically labeled internal standards has proven to be a successful approach in that regard, but has yet to reach its full potential in terms of multiplexing and sensitivity. Here, we report the development of a new MRM method for the quantitation of 253 disease-associated proteins (represented by 625 interference-free peptides) in 13 LC fractions. This 2D RPLC/MRM-MS approach extends the depth and breadth of the assay by 2 orders of magnitude over pre-fractionation-free assays, with 31 proteins below 10 ng/mL and 41 proteins above 10 ng/mL now quantifiable. Standard flow rates are used in both chromatographic dimensions, and up-front depletion or antibody-based enrichment is not required. The LC separations utilize high and low pH conditions, with the former employing an ammonium hydroxide-based eluent, instead of the conventional ammonium formate, resulting in improved LC column lifetime and performance. The high sensitivity (determined concentration range: 15 mg/mL to 452 pg/mL) and robustness afforded by this method makes the full MRM panel, or subsets thereof, useful for the verification of disease-associated plasma protein biomarkers in patient samples.

Biological significance: The described research extends the breadth and depth of protein quantitation in undepleted and non-enriched human plasma by employing standard-flow 2D RPLC/MRM-MS in conjunction with a complex mixture of isotopically labeled peptide standards. The proteins quantified are mainly putative biomarkers of non-communicable (i.e., non-infectious) disease (e.g., cardiovascular or cancer), which require pre-clinical verification and validation before clinical implementation. Based on the enhanced sensitivity and multiplexing, this quantitative plasma proteomic method should prove useful in future candidate biomarker verification studies.