Amplify, Amplify: Shotgun Proteomics Boosts the Signal for Biomarker Discovery

Abstract

Disease processes or exposure to environmental toxicants can produce tiny modifications (called adducts) on proteins in the blood. A clinical assay that reliably detects those modifications in plasma or serum could confirm environmental exposures or speed diagnosis of diseases such as cancer. But scientists have identified so many protein adducts that might serve as candidate biomarkers that finding the best ones to advance into further testing presents a major challenge. Advancements in an approach known as shotgun proteomics now promise to streamline the discovery process by identifying the most promising biomarkers for exploration. Scientists are still laying the rudiments of a systematic “pipeline” for biomarker discovery, says Dan Liebler, a professor of biochemistry, pharmacology, and biomedical informatics at Vanderbilt University School of Medicine. Liebler has for almost 10 years used proteomics to study protein damage caused by oxidative stress or chemical toxicity induced by reactive endogenous chemicals. “We have knowledge of [reactions or changes] that could be advanced at some point to biomarkers, but there are so many candidate changes in tissues or living systems exposed to environmental stressors, and we don’t have efficient mechanisms for identifying the best possible markers to move forward in the pipeline,” Liebler says. In the last 10 years proteomics technologies involving mass spectrometry have made it possible to quickly identify proteins and quantify adducts, down to the very amino acid site of modification, so scientists can more quickly screen potential biomarkers. In a typical shotgun proteomics approach, a scientist would take a biologic sample, add enzymes to digest all the proteins to peptides, fractionate the peptides, then analyze them on an ion trap mass spectrometer. The resulting spectra can be compared against peptide databases to determine which proteins are present in the sample. Scientists have used the shotgun pro−teomics approach to discover many protein modifications of interest. But the list of biomarker candidates must be narrowed by determining which ones can be reproducibly measured in large numbers of clinical samples, such as blood samples from unexposed and exposed people. Performing such tests of candidate bio−markers currently requires development of targeted immunoassays, which is difficult and expensive because scientists have to develop an antibody for use in the assay that is specific to the protein of interest. Improvements in mass spectrometry assays have the potential to change that. Proteomics researchers now predict that in as little as three to four years hybrid immuno–mass spectrometry assays, which combine some elements of traditional immunoassays with some elements of shotgun proteomics, will be used to evaluate candidate bio−markers in a large number of samples.