Enhanced sensitivity and multiplexing with 2D LC/MRM-MS and labeled standards for deeper and more comprehensive protein quantitation.
Mass spectrometry (MS)-based protein quantitation is increasingly being employed to verify candidate protein biomarkers. Multiple or selected reaction monitoring-mass spectrometry (MRM-MS or SRM-MS) with isotopically labeled internal standards has proven to be a successful approach in that regard, but has yet to reach its full potential in terms of multiplexing and sensitivity. Here, we report the development of a new MRM method for the quantitation of 253 disease-associated proteins (represented by 625 interference-free peptides) in 13 LC fractions. This 2D RPLC/MRM-MS approach extends the depth and breadth of the assay by 2 orders of magnitude over pre-fractionation-free assays, with 31 proteins below 10 ng/mL and 41 proteins above 10 ng/mL now quantifiable. Standard flow rates are used in both chromatographic dimensions, and up-front depletion or antibody-based enrichment is not required. The LC separations utilize high and low pH conditions, with the former employing an ammonium hydroxide-based eluent, instead of the conventional ammonium formate, resulting in improved LC column lifetime and performance. The high sensitivity (determined concentration range: 15 mg/mL to 452 pg/mL) and robustness afforded by this method makes the full MRM panel, or subsets thereof, useful for the verification of disease-associated plasma protein biomarkers in patient samples.


CONCLUSIONS
The described research extends the breadth and depth of protein quantitation in undepleted and non-enriched human plasma by employing standard-flow 2D RPLC/MRM-MS in conjunction with a complex mixture of isotopically labeled peptide standards. The proteins quantified are mainly putative biomarkers of non-communicable (i.e., non-infectious) disease (e.g., cardiovascular or cancer), which require pre-clinical verification and validation before clinical implementation. Based on the enhanced sensitivity and multiplexing, this quantitative plasma proteomic method should prove useful in future candidate biomarker verification studies.
University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, BC V8Z 7X8, Canada.
Andrew J
AJ
Percy
University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, BC V8Z 7X8, Canada.
Romain
R
Simon
University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, BC V8Z 7X8, Canada.
Andrew G
AG
Chambers
University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, BC V8Z 7X8, Canada; Department of Biochemistry and Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Rd., Victoria, BC V8P 5C2, Canada. Electronic address: christoph@proteincentre.com.
Christoph H
CH
Borchers
D064753:Ammonium Hydroxide / Q000737:chemistry; D000906:Antibodies / Q000737:chemistry; D015415:Biomarkers / Q000737:chemistry; D001798:Blood Proteins / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay; D013058:Mass Spectrometry / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry; D040901:Proteomics / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity
D000906:Antibodies; D015415:Biomarkers; D001798:Blood Proteins; D010446:Peptide Fragments; D064753:Ammonium Hydroxide
Fractionation; MRM; Multiple reaction monitoring; Plasma; Protein quantitation; Two-dimensional chromatographic separation