Quantitative Proteomics



Activities:

MS-based protein quantitation is performed to determine differences between samples from healthy and diseased patients in biomarker and systems biology studies. At the Centre, relative quantitation (via iTRAQ) is conducted to obtain fold changes of abundance levels in protein biomarker discovery studies, while absolute quantitation (by MRM with our well characterized and concentration balanced isotopically labeled peptides as internal normalizers) is used to provide exact endogenous concentrations for protein biomarker verification/validation. The impetus lies in protein biomarker screening for earlier detection and improved patient outcomes.

Further to our absolute quantitation capabilities, robust methods continue to be developed for the highly multiplexed and sensitive quantitation of putative protein disease biomarkers in a variety of biological samples. In our most recent efforts, we have quantified 192, 136, 130, and 96 endogenous proteins (all spanning at least 5 orders of magnitude in concentration) in plasma, urine, cerebrospinal fluid, and dried blood spots, respectively. While these methods are antibody-free, enhanced detection sensitivity (down to the mid pg/mL range in human plasma) has been achieved with 2D RPLC-MRM/MS, operated at standard-flow rates and under alkaline/acidic mobile phase conditions. All methods are rigorously assessed and qualified before implementation in small- and large-scale proteomic studies for improved precision and accuracy. Central to this is our in-house developed standardization kits, which are designed for quality control of all analytical processes.


Methods:

Relative Quantitation
immunodepletion and bottom-up 2D LC-MS
→ iTRAQ 8-plex
→ standard-flow high pH RPLC and nano-flow low pH LC-MS/MS
→ Orbitrap Fusion Tribrid

Absolute Quantitation
bottom-up 1D and 2D LC-MRM/MS with complex SIS peptide mixtures
→ standard-flow RPLC separations
→ MRM on QqQ mass spectrometers (Agilent’s 6495 and 6490)



Results Highlights:

Multiplexed MRM with Internal Standards for Cerebrospinal Fluid Candidate Protein Biomarker Quantitation Percy AJ, Yang J, Chambers AG, Simon R, Hardie DB, Borchers CH J Proteome Res. 2014 Jun 30 PMID: 24911472 http://www.ncbi.nlm.nih.gov/pubmed/24911472

Enhanced sensitivity and multiplexing with 2D LC/MRM-MS and labeled standards for deeper and more comprehensive protein quantitation. J Proteomics. 2014 Jun 25;106:113-24. doi: 10.1016/j.jprot.2014.04.024. Epub 2014 Apr 24 Percy AJ, Simon R, Chambers AG, Borchers CH http://www.ncbi.nlm.nih.gov/pubmed/24769237

Method and platform standardization in MRM-based quantitative plasma proteomics. J Proteomics. 2013 Dec 16;95:66-76. doi: 10.1016/j.jprot.2013.07.026. Epub 2013 Aug 7 Percy AJ, Chambers AG, Yang J, Jackson AM, Domanski D, Burkhart J, Sickmann A, Borchers CH http://www.ncbi.nlm.nih.gov/pubmed/23933160

Advances in multiplexed MRM-based protein biomarker quantitation toward clinical utility. Biochim Biophys Acta. 2014 May;1844(5):917-26. doi: 10.1016/j.bbapap.2013.06.008. Epub 2013 Jun 25 Percy AJ, Chambers AG, Yang J, Hardie DB, Borchers CH http://www.ncbi.nlm.nih.gov/pubmed/23806606

Multiplexed quantitation of endogenous proteins in dried blood spots by multiple reaction monitoring-mass spectrometry. Mol Cell Proteomics. 2013 Mar;12(3):781-91. doi: 10.1074/mcp.M112.022442. Epub 2012 Dec 7. Chambers AG, Percy AJ, Yang J, Camenzind AG, Borchers CH http://www.ncbi.nlm.nih.gov/pubmed/23221968

MRM-based multiplexed quantitation of 67 putative cardiovascular disease biomarkers in human plasma. Proteomics. 2012 Apr;12(8):1222-43. doi: 10.1002/pmic.201100568. Domanski D, Percy AJ, Yang J, Chambers AG, Hill JS, Freue GV, Borchers CH http://www.ncbi.nlm.nih.gov/pubmed/22577024

Comparison of standard- and nano-flow liquid chromatography platforms for MRM-based quantitation of putative plasma biomarker proteins. Anal Bioanal Chem. 2012 Sep;404(4):1089-101. doi: 10.1007/s00216-012-6010-y. Epub 2012 May 1 Percy AJ, Chambers AG, Yang J, Domanski D, Borchers CH http://www.ncbi.nlm.nih.gov/pubmed/22547352

Assay development for the determination of phosphorylation stoichiometry using multiple reaction monitoring methods with and without phosphatase treatment: application to breast cancer signaling pathways. Anal Chem. 2010 Jul 1;82(13):5610-20. doi: 10.1021/ac1005553. Domanski D, Murphy LC, Borchers CH http://www.ncbi.nlm.nih.gov/pubmed/20524616


Current Members:

Andrew Chambers, PhD
Juncong Yang
Darryl Hardie
Nicole Sessler, PhD
Monica Elliott
Angela Jackson