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Conference shines light on latest in mass spec

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Y. Victoria Zhang, PhD, MBA

February 2019—Mass spectrometry has been proved to be an essential and powerful platform for clinical diagnostics. The value it adds to health care has increased steadily in the past decade, as was showcased at the 8th Annual American Association for Clinical Chemistry Conference on Mass Spectrometry and Separation Sciences for Laboratory Medicine, held last fall in Philadelphia. Stakeholders from academia, hospitals, industry, and regulatory bodies came together to exchange information about developments in the technology and best practices to meet patient care needs.

Dr. Zhang

Pre-conference on-site workshops helped attendees gain firsthand experience with and knowledge of mass spectrometry’s applications in clinical care and omics-based research functions. The University of Pennsylvania, Children’s Hospital of Philadelphia, Temple University, and NMS Labs opened their doors to host the events.

The conference consisted of 18 presentations, among them three by young investigators, and one panel discussion. Here are highlights of six of the 18 presentations.

Dennis J. Dietzen, PhD, AACC president and professor of pathology and immunology at Washington University in St. Louis, gave a plenary lecture in which he spoke about the utility of traditional metabolic profiling in pediatric and maternal-fetal pathology. He asked a key question: While these metabolic profiles are used routinely to detect inborn errors of metabolism such as PKU and MCAD deficiency, can they shed light on other disorders with unclear pathogenesis? When considered in the proper clinical context, he said, circulating concentrations of amino acids and carnitine esters provide a wealth of information about nutrition, protein synthesis, metabolic fuel utilization, vascular tone, and nitrogen balance.

Dr. Dietzen talked about animal and human studies to uncover metabolic patterns that might predict liver regeneration, poor pregnancy outcome, and severe neonatal hypoglycemia. Circulating lysine and methionine catabolites reflect liver regeneration and suggest a better prognosis in children with acute liver failure. Such markers of liver regeneration may augment the common biomarkers of hepatocyte dysfunction, such as bilirubin, albumin, and prothrombin time. Elevated maternal concentrations of arginine, alanine, hydroxyproline, and glutamate in the first trimester appear to predict the occurrence of preeclampsia and intrauterine growth retardation. Finally, increased concentrations of acetyl carnitine and branched chain amino acids in neonates characterize a poor ketogenic response to neonatal hypoglycemia and may identify infants who need therapeutic dietary supplementation. The receiver operating characteristics for these profiles yielded areas from 0.75 to 0.85, suggesting that subtle metabolic alterations in these case/control cohorts can provide diagnostic and mechanistic insight to maternal-fetal pathology. Further studies may clarify the metabolic basis of these childhood disorders and lead to more precise prediction, identification, and treatment.

Neil L. Kelleher, PhD, Walter and Mary E. Glass professor of molecular biosciences, professor of chemistry and medicine, and director of the Proteomics Center of Excellence at Northwestern University Feinberg School of Medicine, presented the increasingly important role of proteoforms in human health and disease. The post-translational modification (PTM) of proteins is a critical method for regulating protein activity, he said, but typical proteomic approaches to identifying and localizing PTMs require enzymatic digestion of proteins to generate peptides. These bottom-up proteomic approaches can be useful in high-throughput identification of peptide PTMs, but they suffer from the protein inference problem and are further complicated when amino acid substitutions and multiple possible PTMs can be colocalized onto the same protein molecule.

Top-down proteomics (TDP) circumvents the protein inference problem by identifying the full-length, undigested protein molecule complete with possible amino acid substitutions and PTMs, and these protein forms are called proteoforms. Consequently, top-down proteomics can be used to better understand health and disease by providing a more comprehensive view of the functional proteins encoded by the 20,000 human genes. With top-down proteomics, Dr. Kelleher’s group has mapped 11 proteoforms from the oncogene KRAS in the context of colorectal cancer (Ntai I, et al. Proc Natl Acad Sci U S A. 2018;115[16]:​4140–4145). And they have begun to understand how driving mutations in the KRAS gene affect post-translational modifications elsewhere on the expressed protein in a low-bias and isoform-specific manner (i.e. KRAS4b versus KRAS4a).

Furthermore, acute organ rejection after an organ transplant is difficult to predict and even more challenging to detect before acute rejection and organ failure. Dr. Kelleher and colleagues used top-down proteomics to identify proteoforms that could serve as potential biomarkers for acute liver rejection in a pilot study of patients who were transplant excellent or had undergone acute rejection.

Together, these studies have underscored the central role of a proteoform-centric approach when studying human health and disease, and these cases demonstrate the need for the proteomics community to consider making the switch from a peptide-centric approach to protein- and proteoform-based approaches.

Henry Rodriguez, PhD, MS, MBA, director of the Office of Cancer Clinical Proteomics Research and deputy director (acting) of the Center for Strategic Scientific Initiatives, National Cancer Institute, discussed the convergence of proteomics and genomics, or proteogenomics, and how this emerging, multidisciplinary field is advancing basic and translational cancer research. He provided a look at the NCI’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) and discussed its roots in addressing analytical rigor and reproducibility in proteomic technologies, which led to the standardization of untargeted (discovery-based) and targeted (confirmatory and clinical-based) protein analyses by mass spectrometry, production of quality reagents and resources (antibodies and assays), and development of community-based proteomic open-data sharing policies.

CPTAC’s initial pilot studies on genomically characterized colorectal (Zhang B, et al. Nature. 2014;513​[7518]:​382–387), breast (Mertins P, et al. Nature. 2016;534[7605]:55–62), and ovarian (Zhang H, et al. Cell. 2016;​166[3]:​755–765) tumors from The Cancer Genome Atlas revealed new insights into the molecular subtypes of cancers that could not be uncovered with next-generation sequencing only. The data, published in three flagship papers, indicate the usefulness of a proteogenomic approach in providing a new paradigm for understanding cancer biology. In its current iteration, CPTAC has two sub-programs. The tumor characterization program uses standardized and harmonized proteomic workflows to further characterize additional cancer types, and the translational research program addresses questions of biology in the context of NCI-sponsored clinical trials.

Dr. Rodriguez highlighted the growing interest in applying proteogenomics to precision oncology. CPTAC has forged partnerships with other federal and international entities to generate multimodal data, accelerate progress in cancer research, ensure greater cooperation and collaboration, share data with the greater research community, and ultimately bridge the gap between cancer genomics and clinical action.

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