Shining Targeted Proteomics Solutions: Precision, Robustness and Throughput

Determining calibration curves for robust, targeted, and high throughput proteomics with an Evosep One and Agilent 6495C LC/TQ

When transitioning from discovery proteomics to targeted protein quantification, the multiple reaction monitoring (MRM)-based LC/MS method plays an essential role. Sample complexity and the low concentration of certain proteins are the major challenges encountered when performing protein analysis using targeted methods. Recent advancements in low-flow HPLC and acquisition rates in QQQ instrumentation is enabling MRM-based analytical methods using stable isotope-labeled standard (SIS) peptides for targeted quantitative proteomics to improve sensitivity, linear range, precision, accuracy, robustness, and most importantly throughput in the data acquisition as well as simplified data processing. Using a commercially available complex proteomics sample (PeptiQuant Plus 125 from MRM Proteomics) spiked in a plasma matrix, calibration curves were established highlighting the significant quantitative advantages of MRM-based targeted quantification. In addition, a select number of peptides from the complex proteomics sample were quantified with very high productivity methods combining the use of Evosep One and Agilent 6495C LC/TQ. Quantification with 3- and 5-min low-flow gradients (0.15 mm ID column) and 4-5 min re-equilibrations are demonstrated with short dwell times offering excellent quantitative performance with high throughput. Sensitivity, linearity, and precision is demonstrated with these fast low-flow methods demonstrating inject-to-inject cycle times under 10 minutes. This study demonstrated the suitability of this new high-performance LC/MS system configuration for high throughput protein quantification, even with very limited sample amount.

Presenter: Wendi Hale (LC/MS Applications Scientist)

Wendi has worked at Agilent for four and a half years as an LC/MS Applications Scientist.  She specializes in intact protein analysis, peptide mapping, and proteomics.  She received her Ph.D. in Analytical Chemistry from the University of Michigan, developing methods for biomolecule analysis with LC/FT-ICR MS. She completed her post-doctoral work at the University of Geneva, establishing phosphoproteomics in a molecular biology lab studying TOR signaling pathways.

Hitting the Achilles' Heel of Cancers with Confidence - Robust Validation of Neoantigens

Proteins encoded by mutant genes in cancers can be processed and presented on tumor cell surface by human leukocyte antigen (HLA) molecules, and such mutant peptides are called Neoantigens. Neoantigens are naturally existing tumor markers. To identify the exact sequence of a neoantigen with high confidence is the key to the success of many cancer immunotherapeutics. In this presentation, we will discuss advancements and difficulties of prediction-dependent and prediction-independent neoantigen identification strategies. We will review the latest development of technologies for neoantigen identification and introduce our unique neoantigen detection and quantification pipeline based on a triple quadrupole mass spectrometer.

Learning Objectives:

• Gain familiarity of the advancements and difficulties of prediction-dependent and prediction-independent neoantigen identification strategies.
• Understand the latest development of technologies for neoantigen identification

Presenter: Qing Wang, PhD (MHS, MS - Founder and CEO of Complete Omics Inc.)

Dr. Wang graduated from Johns Hopkins University with a PhD, supervised by Dr. Bert Vogelstein at the School of Medicine, and an MHS in Biostatistics, supervised by Dr. Rafael Irizarry at the Bloomberg School of Public Health. Before coming to the US, he obtained his BS and MS in Molecular Biology from Nankai University and trained with Dr. Tianhui Zhu, the Founding Dean of Nankai University School of Medicine. Dr. Wang is also currently pursuing an Executive MBA from The Wharton School of the University of Pennsylvania and will obtain his degree in 2023. During the past 15 years, Dr. Wang's research has focused on clinical proteomics and clinical genomics. Through Complete Omics Inc., Dr. Wang is working to break the boundaries between different fields in medicine and the life sciences, and to comprehensively leverage the features of omics technologies to achieve improved personalized disease management and precision medicine.ounder and CEO of Complete Omics Inc.

Quantitative Proteomics in Biochemical and Cancer Research

Protein biomarker study usually includes the transition from untargeted proteomics for the discovery of biomarkers to targeted proteomics for validation and absolute quantitation. Proteomics typically involves “bottom up” proteomics – the analysis of peptides resulting from the digestion of the parent proteins. Untargeted (“shotgun”) proteomics can provide relative quantitation, i.e. up- or down- regulation of proteins or pathways. The actual concentrations (e.g., ng/mL) of these proteins in human samples can be determined by using methods specifically targeting a smaller number of proteins. The “gold-standard” targeted-proteomics method is multiple reaction monitoring mass spectrometry (MRM-MS), a technique involving detection of a specific precursor ion and a specific fragment ion, typically with on-line LC separation and stable-isotope-labeled (SIS) peptides which compensate for any changes in instrument performance, sensitivity, or LC retention time. MRM-MS has been found to provide accurate and reproducible quantitation across multiple laboratories. My laboratory has already developed ~6000 MRM assays for proteins from plasma and other biofluids (e.g., CSF and urine), in humans and other species. Customized combinations of these assays can be used to create multiplexed MRM panels, targeting proteins which have been found to be biomarkers of a specific disease. These panels can also be turned into kits which contain enzymes, standards, SIS peptides, and instrument parameters. Proteogenomics involves the combination of proteomics and genomics. While genomics involves the potential for the expression of a particular protein, proteomics can tell us whether this protein is actually expressed, as well as its phosphorylation status. In a recent proteogenomics study on metastatic colorectal cancer tumours, we detected more than 9,000 proteins using shotgun proteomics, and found expression changes in numerous proteins involved in cancer progression and resistance, as well as peptides representing the predicted somatic mutations, including KRASG12V. A targeted MS assay for 8 mutated peptides allowed quantitative mutation-level phenotyping. One tumour sample was found to have a phenotype that was 90% KRASwildtype and only 10% KRASG12V. Based on hotspot sequencing, this tumour sample would not be expected to respond to anti-EGFR treatment. MS-based tumour-phenotyping, however, indicated that this tumour might respond to anti-EGFR treatment.

Presenter: Christoph Borchers, PhD. (Professor at McGill University, Department of Oncology)

Professor in the Department of Oncology at McGill University, and holds the Segal Chair in Molecular Oncology. His research involves proteomics and metabolomics technologies for clinical diagnostics and structural proteomics. He is also head of a newly established Omics laboratory for personalized medicine and health at Skoltech, Moscow, Russia.