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.

Multiplexed targeted mass spectrometry assays for prostate cancer-associated urinary proteins

“…AGR2, AGR3, CCL3, CEAM5, and CEAM6 were reliably detected and quantified except CCL4 and WISP1 (Figure 1 ). The reproducibility of LG-SRM and PRISM-SRM based assays for measurements in biofluids such as urine and serum was well validated in our previous reports, which typically had a coefficient of variance (CV) <10% [ 16 , 17 , 20 ]. With a combined LG-SRM and PRISM-SRM, SRM assays were established for each of the detectable peptides: three best transitions without matrix interference and the best transition for quantitation (Table 1 ).…”

Clinical potential of mass spectrometry-based proteogenomics

“…Additionally, while many proteins and posttranslational modifications of high clinical interest will be detectable in clinical samples by immuno-MRM, some are likely to remain below the lower limit of quantification of the assay. In those cases where a sufficiently high affinity anti-peptide antibody cannot be developed, there are alternative approaches involving extensive sample fractionation that also provide highly sensitive, reproducible results 127–129 , albeit at lower throughput using high complexity protocols. Second, subcellular localization or spatial resolution of protein expression (e.g., in different regions of a tumor with clonal heterogeneity, invasive edge vs hypoxic center of tumor) is not possible without additional sample processing such as macro- or micro-dissection of biospecimens.…”

Mass spectrometry in drug metabolism and pharmacokinetics: Current trends and future perspectives

“…The main reason for this might be found in the preconcentration of matrix compounds that thereby induces additional ion suppression. This can be overcome by applying offline or online 2D‐LC approaches using a large ID 1 st dimension column allowing the injection of a relatively high sample volume and a small ID 2 nd dimension column to maximize sensitivity. The offline approach allows easier analysis of multiple analytes, while the online heart cut 2D‐LC approach lowers the risk of compound loss due to recovery and/or degradation issues.…”