Sensitive detection of low-abundance proteins in complex biological samples has typically been achieved by immunoassays that use antibodies specific to target proteins; however, de novo development of antibodies is associated with high costs, long development lead times, and high failure rates. To address these challenges, we developed an antibody-free strategy that involves PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing) for sensitive selected reaction monitoring (SRM)-based targeted protein quantification. The strategy capitalizes on high-resolution reversed-phase liquid chromatographic separations for analyte enrichment, intelligent selection of target fractions via on-line SRM monitoring of internal standards, and fraction multiplexing before nano-liquid chromatography-SRM quantification. Application of this strategy to human plasma/serum demonstrated accurate and reproducible quantification of proteins at concentrations in the 50-100 pg/mL range, which represents a major advance in the sensitivity of targeted protein quantification without the need for specific-affinity reagents. Application to a set of clinical serum samples illustrated an excellent correlation between the results obtained from the PRISM-SRM assay and those from clinical immunoassay for the prostate-specific antigen level.biomarker verification | systems biology | targeted proteomics S elected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), has recently emerged as a promising technology (1-16) for high-throughput mass spectrometry (MS)-based quantification of targeted proteins in biological and clinical specimens (e.g., tumor tissues). SRM has demonstrated relatively good selectivity, reproducibility (or precision), and sensitivity for a range of multiplexed protein assays (2, 4-8, 11, 17, 18) and has potential for quantifying protein isoforms (19) and posttranslational modifications (PTMs) (3,(20)(21)(22) for which good quality antibodies often do not exist. Nevertheless, a major limitation of current SRM technology is insufficient sensitivity for detecting low-abundance proteins present at sub-nanogram per milliliter levels in human blood plasma or serum and extremely low-abundance proteins in cells or tissues. Without sample prefractionation, liquid chromatography (LC)-SRM measurements have been limited to only moderately abundant proteins in human plasma present in the low microgram per milliliter range (2,6,8).More recently, the combination of immunoaffinity depletion and fractionation by strong cation exchange (SCX) chromatography (4, 23), along with advances in MS sensitivity (24), has extended SRM quantification of plasma proteins to low nanogram per milliliter levels (4, 23). SISCAPA (stable isotope standards and capture by anti-peptide antibodies) coupled with SRM demonstrated quantification of target proteins in the same range, using as little as 10 μL of human plasma (8,(25)(26)(27). SISCAPA assays have some distinct advantages over conventiona...

Mass spectrometry (MS)-based high-throughput proteomics is the core technique for large-scale protein characterization. Due to the extreme complexity of proteomes, sophisticated separation techniques and advanced MS instrumentation have been developed to extend coverage and enhance dynamic range and sensitivity. In this review, we discuss the separation and prefractionation techniques applied for large-scale analysis in both bottom-up (i.e., peptide-level) and top-down (i.e., protein-level) proteomics. Different approaches for quantifying peptides or intact proteins, including label-free and stable-isotope-labeling strategies, are also discussed. In addition, we present a brief overview of different types of mass analyzers and fragmentation techniques as well as selected emerging techniques.

Characterization of the mature protein complement in cells is crucial for a better understanding of cellular processes on a systems-wide scale. Toward this end, we used single-dimension ultra–high-pressure liquid chromatography mass spectrometry to investigate the comprehensive “intact” proteome of the Gram-negative bacterial pathogen Salmonella Typhimurium. Top-down proteomics analysis revealed 563 unique proteins including 1,665 proteoforms generated by posttranslational modifications (PTMs), representing the largest microbial top-down dataset reported to date. We confirmed many previously recognized aspects of Salmonella biology and bacterial PTMs, and our analysis also revealed several additional biological insights. Of particular interest was differential utilization of the protein S-thiolation forms S-glutathionylation and S-cysteinylation in response to infection-like conditions versus basal conditions. This finding of a S-glutathionylation-to-S-cysteinylation switch in a condition-specific manner was corroborated by bottom-up proteomics data and further by changes in corresponding biosynthetic pathways under infection-like conditions and during actual infection of host cells. This differential utilization highlights underlying metabolic mechanisms that modulate changes in cellular signaling, and represents a report of S-cysteinylation in Gram-negative bacteria. Additionally, the functional relevance of these PTMs was supported by protein structure and gene deletion analyses. The demonstrated utility of our simple proteome-wide intact protein level measurement strategy for gaining biological insight should promote broader adoption and applications of top-down proteomics approaches.

When transcription from the human U6 snRNA gene is reconstituted with recombinant factors and purified RNA polymerase III (pol III), pol III must be treated with CK2 to be active. We show that highly purified pol III contains associated beta-actin, and beta-actin localizes to an active U6 promoter in vivo. Pol III immunoprecipitated from IMR90 cells treated with a genotoxic agent lacks associated beta-actin and is inactive in the reconstituted assay. Transcription is regained upon treatment of pol III with CK2 and addition of beta-actin. This suggests that beta-actin associated with pol III is essential for basal pol III transcription.