Multiple Reaction Monitoring Cubed for Protein Quantification at the Low Nanogram/Milliliter Level in Nondepleted Human Serum

Abstract

Mass spectrometry-based strategies for the quantification of low-abundance putative protein biomarkers in human blood currently require extensive sample fractionation steps which hamper their implementation in a routine and robust way across clinical laboratories. We demonstrate that a technique using MS(3) reconstructed chromatograms on a signature of secondary ions issued from a trapped primary product ion, termed multiple reaction monitoring cubed (MRM(3)), enables targeting protein biomarkers in the low nanogram/milliliter range in nondepleted human serum. The simple two-step workflow is based on a trypsin proteolysis of whole serum (100 microL) followed by enrichment of targeted proteotypic peptides on a solid phase extraction column using mixed-cation exchange resin. MRM(3)'s fidelity of peak detection extends the dynamic range and limit of quantitation (LOQ) of protein biomarkers to the low nanogram/milliliter range, corresponding to a concentration that is 10(6)-fold lower than the concentration of the most abundant proteins in serum. The power of the MRM(3) method is illustrated by the assay of prostate specific antigen in nondepleted human sera of patients. The results correlate well with the established method for determining PSA levels in serum, i.e., enzyme-linked immunosorbent assay (ELISA) tests.

Proteomics of Saccharomyces cerevisiae Organelles

“…One could envision that such an experiment would use a multidimensional fractionation scheme and extensive analysis of co-fractionation profiles that follow from quantitative mass spectrometry using e.g. the multiple reaction monitoring method (57,58). We expect that combining these data with those from co-purification strategies where protein complexes are isolated using tagged proteins will provide a more complete and accurate description of protein localization.…”

Mass Spectrometry for Biomarker Development

“…However, for SRM methods to approach the performance of immunoassays for measurement of low-abundance protein biomarkers, both the sensitivity and dynamic range of SRM measurements need to be further significantly increased. In order to further increase the detection sensitivity of SRM, several important developments have been made to both the front-end separation/enrichment and back-end MS detection, such as stable isotope standards and capture by anti-peptide antibodies (SISCAPA) (Anderson et al 2004); high-pressure, high-resolution separations coupled with intelligent selection and multiplexing (PRISM) (Shi et al 2012a); and selected reaction monitoring cubed (SRM 3 ) (Fortin et al 2009). Effectively reducing sample complexity and matrix interference is critical in order to increase the sensitivity of SRM.…”

Quantitative Clinical Chemistry Proteomics (qCCP) using mass spectrometry: general characteristics and application

“…These systems permit the specific quantitation of low abundant ions through an additional ' MS 3 ' fragmentation step in the ion trap. This approach in has been used to quantify prostate specific antigen (PSA) in non-depleted human serum with satisfactory linearity (10 -1000 μ g/L), sensitivity, and selecti vity [64 ].…”