Laura McIntosh (Presenter)
Caprion Proteome
Bio: Laura is Vice President, Translational Research at Caprion and is responsible for the scientific management of proteomics studies. She has been integrally involved in a personalized medicine initiative in the province of Quebec, which brings together academic and pharmaceutical partners, and has been the champion for the use of mass spectrometry-based approaches. Prior to joining Caprion, Laura was President and General Manager of Osprey Pharmaceuticals, where she oversaw the development of a platform of therapeutic proteins for the modulation of inflammation. Her previous experience also includes positions at ART Advanced Research Technologies and Argose Inc, where she was involved in the development of novel optimal imaging technologies. Laura earned a PhD in Cell Biology from the University of Manitoba, followed by a post-doctoral fellowship at the National Research Council of Canada.
Authorship: L.McIntosh(1), G.Batist (2,3), A.Manceur(1), V.Brechler(1), S.Parent(1), M.Schirm(1), A.Jang(1), R.Guilbaud(1), N.Guigal-Stephan(4), B.Lockhart(4) and L.Di Donato(1)
1)Caprion Proteome, Montreal, QC; (2)Segal Cancer Centre-Jewish General Hospital, Montreal, QC; (3)Quebec Clinical Research Organization in Cancer, Montreal, QC; (4) Servier, Croissy-sur-seine, France
| Short Abstract LC-MS/MS is a powerful tool for the discovery and quantification of biomarkers. Two case studies will be presented. In the first study, an unbiased approach was used to discover plasma proteins from patients undergoing treatment for metastatic colorectal cancer (mCRC). 1723 unique proteins were identified, and 235 were differentially-expressed between Intrinsic Resistance and Responder groups at baseline. In the second study, an MRM assay for the absolute quantitation of 9 cancer-related proteins (55 unique peptides) in cell lysates was developed. The LLOQ was ≤ 10 ng/mL and good precision (< 30%) and accuracy (70-130%) was achieved. Both LC-MS/MS methods have an important role in the advancement of personalized medicine. |
Long Abstract
Introduction: The discovery and validation of novel biomarkers of resistance or response to therapy is a critical component for the advancement of personalized medicine. Liquid chromatography mass spectrometry (LC-MS/MS) has enormous potential for the identification of protein candidates in biological samples and subsequent validation using multiple reaction monitoring (MRM). Two case studies demonstrating the power of MS for discovery and quantification of novel biomarkers for the prediction of response to drug treatment are presented.
Methods: Discovery study: An unbiased label-free, gel-free MS approach was used to profile proteins present in plasma from patients undergoing treatment for metastatic colorectal cancer (mCRC). Baseline samples were taken, as well as samples at approximately 2 and 7 months post-treatment with first-line chemotherapy (XOLOX or FOLFOX + Bevaciaumab). Patients were grouped into Responders, Intrinsic and Acquired Resistance groups. Samples were depleted with IgY14/Supermix, digested with trypsin and fractionated with strong cation exchange, prior to injection onto a Q- ExactiveTM mass spectrometer. The raw mass spectrometer data files for each LC-MS run for each fraction were aligned independently using Elucidator (Rosetta Biosoftware). Peak intensities for each detected isotope group were then extracted across all samples. The MS/MS spectra were then matched to the corresponding peptide sequences found in the UniProt human protein database using Mascot software. A statistical analysis was performed at the peptide and protein-level to assess differential expression. Absolute Quantitation: A multiplexed multiple reaction monitoring (MRM) assay for the measurement of 9 cancer-related proteins (55 unique tryptic peptides) in cell lysates was developed. To compensate for analytical variability, tryptic heavy-labeled winged peptides were added to all samples at a fixed concentration during processing. Recombinant proteins were used for the preparation of calibration standards and Quality Control (QC) samples. Samples were injected onto a NanoAcquity UPLC (Waters) coupled to a QTRAP6500 mass spectrometer (AB Sciex). Assay development consisted of testing for sensitivity, specificity, establishing the curve fit, concentration of heavy-labeled peptides, curve range, and evaluation of matrix effect, carry-over and precision and accuracy.
Results: Discovery study: 1723 unique proteins were identified using the discovery data, with 235 proteins being differentially-expressed between the Intrinsic Resistance and Responder groups at baseline. In addition, 141 proteins were differentially expressed (i.e., changed over time) in the Acquired Resistance group. The differentially expressed proteins had associations with gastrointestinal cancer and cell adhesion and migration processes and metastasis. A list of 300 proteins has been assembled and will be used to validate the biomarkers in an independent cohort of patients using MRM. Absolute Quantitation: Eight of the nine proteins in the MRM assay had an LLOQ ≤ 10 ng/mL. At least 1 peptide per protein had a precision of < 30% in the curve range. Accuracies were within 70-130% for ≥ 75% of the standards and ≥ 67% of QC samples. Endogenous protein levels ranged from 0.5 – 169 pg/µg.
Conclusions: We demonstrate the power of a non-biased approach for the discovery of blood-based biomarkers that can differentiate patients that respond to those that have resistance to treatment in first-line mCRC. Additionally, we show that MRM can precisely and accurately quantify tumor biomarkers. Ultimately, these data have the potential to guide treatment regimens and predict drug efficacy, leading to new avenues for personalized therapies for cancer treatment.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Caprion Proteome |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |