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 two-step 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, PROMIS-Quan 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.
Highlights
Biomarker discovery in plasma is one of the holy grails of the proteomic field toward the development of noninvasive diagnostic/prognostic tests [1]
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
The abbreviations used are: FDR, false discovery rate; PROMISQuan, PROteomics of MIcroparticles with Super-SILAC Quantification; SILAC, stable isotope labeling with amino acids in cell culture; Cell line microparticles (CLMPs), cell line microparticles; PSA, prostate specific antigen; DMEM, Dulbecco’s Modified Eagle Medium; RPMI, Roswell Park Memorial Institute; FBS, fetal bovine serum; UHPLC, ultra high performance liquid chromatography; FWHM, full width at half-maximum
Summary
Proteomics of Microparticles with SILAC Quantification (PROMIS-Quan): A Novel Proteomic Method for Plasma Biomarker Quantification*□S. The abbreviations used are: FDR, false discovery rate; PROMISQuan, PROteomics of MIcroparticles with Super-SILAC Quantification; SILAC, stable isotope labeling with amino acids in cell culture; CLMP, cell line microparticles; PSA, prostate specific antigen; DMEM, Dulbecco’s Modified Eagle Medium; RPMI, Roswell Park Memorial Institute; FBS, fetal bovine serum; UHPLC, ultra high performance liquid chromatography; FWHM, full width at half-maximum. In the current work we applied state of the art proteomics to study the microparticle proteome and developed the PROteomics of MIcroparticles with Super-SILAC Quantification (PROMIS-Quan) method, which combines deep plasma microparticle coverage of more than 3200 proteins in a single run, with dual-mode relative and absolute Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) quantification. We demonstrated its utilization on samples of prostate cancer patients, and calculated the absolute amount of PSA, a wellknown prostate cancer biomarker
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
