Proteomics of Microparticles with SILAC Quantification (PROMIS-Quan): A Novel Proteomic Method for Plasma Biomarker Quantification*

Abstract

Unbiased proteomic analysis of plasma samples holds the promise to reveal clinically invaluable disease biomarkers. However, the tremendous dynamic range of the plasma proteome has so far hampered the identification of such low abundant markers. To overcome this challenge we analyzed the plasma microparticle proteome, and reached an unprecedented depth of over 3000 plasma proteins in single runs. To add a quantitative dimension, we developed PROMIS-Quan-PROteomics of MIcroparticles with Super-Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) Quantification, a novel mass spectrometry-based technology for plasma microparticle proteome quantification. PROMIS-Quan enables a twostep relative and absolute SILAC quantification. First, plasma microparticle proteomes are quantified relative to a super-SILAC mix composed of cell lines from distinct origins. Next, the absolute amounts of selected proteins of interest are quantified relative to the super-SILAC mix. We applied PROMIS-Quan to prostate cancer and compared plasma microparticle samples of healthy individuals and prostate cancer patients. We identified in total 5374 plasma-microparticle proteins, and revealed a predictive signature of three proteins that were elevated in the patient-derived plasma microparticles. Finally, PROMISQuan enabled determination of the absolute quantitative changes in prostate specific antigen (PSA) upon treatment. We propose PROMIS-Quan as an innovative platform for biomarker discovery, validation, and quantification in both the biomedical research and in the clinical worlds. Biomarker discovery in plasma is one of the holy grails of the proteomic field toward the development of noninvasive diagnostic/prognostic tests (1). To achieve this goal, proteomics necessitates a comprehensive view of the plasma proteome, accurate proteome quantification, combined with relatively short analytical times to enable multiple sample comparisons. However, MS-based biomarker discovery is limited by the vast dynamic range of the plasma, over 11 orders of magnitude (2, 3), which leads to the masking of "tissue leakage" proteins that comprise of potential biomarkers by the core plasma proteins. Two main complementary strategies have been employed to reach identification of low abundance proteins: (i) Targeted proteomics, in which the MS identifies and quantifies only predetermined peptides, thereby circumventing the system's inherent tendency to preferentially detect abundant proteins. This approach is utilized for validation of preselected candidate markers (4 -6). (ii) Plasma fractionation, which biochemically reduces the complexity of the proteomes, and enables discovery of novel biomarkers (7,8).Targeted MS analysis is dominated by the selected reaction monitoring approach, often in combination with antibodybased enrichment of proteins or peptides and stable isotope labeled standards for quantification (9). This approach benefits from the sensitivity and quantitative capabilities of the triple-quadrupole instruments. Its major limitation...

Integrated Microfluidics for Protein Modification Discovery

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Redefining Tissue Crosstalk via Shotgun Proteomic Analyses of Plasma Extracellular Vesicles

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