Deep-Dive Targeted Quantification for Ultrasensitive Analysis of Proteins in Nondepleted Human Blood Plasma/Serum and Tissues.

ABSTRACT: Mass spectrometry-based targeted proteomics (e.g., selected reaction monitoring, SRM) is emerging as an attractive alternative to immunoassays for protein quantification. Recently we have made significant progress in SRM sensitivity for enabling quantification of low nanograms per milliliter to sub-naograms per milliliter level proteins in nondepleted human blood plasma/serum without affinity enrichment. However, precise quantification of extremely low abundance proteins (e.g., ? 100 pg/mL in blood plasma/serum) using targeted proteomics approaches still remains challenging, especially for these samples without available antibodies for enrichment. To address this need, we have developed an antibody-independent deep-dive SRM (DD-SRM) approach that capitalizes on multidimensional high-resolution reversed-phase liquid chromatography (LC) separation for target peptide separation and enrichment combined with precise selection of target peptide fractions of interest, significantly improving SRM sensitivity by ?5 orders of magnitude when compared to conventional LC-SRM. Application of DD-SRM to human serum and tissue provides precise quantification of endogenous proteins at the ?10 pg/mL level in nondepleted serum and at <10 copies per cell level in tissue. Thus, DD-SRM holds great promise for precisely measuring extremely low abundance proteins or protein modifications, especially when high-quality antibodies are not available.

SUBMITTER: Nie S 

PROVIDER: S-EPMC5627997 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature