Protein Biomarker Discovery & Validation

Helping you find the biomarkers that matter

Biomarkers play a critical role in improving the drug development process as well as in the larger biomedical research enterprise.  Scientists look for low abundance proteins found in tissue, blood, urine and other body fluids which may provide vital early indicators of disease. Understanding the relationship between measurable biological processes and clinical outcomes is vital to expanding our arsenal of treatments for all diseases, and for deepening our understanding of normal, healthy physiology.  

Since at least the 1980s, the necessity of using biomarkers as surrogate outcomes in large trials of major diseases, such as cancer and heart disease, has been widely discussed. Biomarkers are significant because they indicate disease onset and/or progression in several therapeutic areas including cancer, neurodegenerative, endocrinology, wellness & nutrition, and cardiovascular research. Discovering biomarkers which point to clear indicators of disease has always been a problematic.  

Mass spectrometry has emerged as a powerful technology for system-wide identification and quantitation of proteins, trace elements and other indicators. LCMS is used routinely for both unbiased discovery-based (untargeted) and targeted determination of changes in protein abundance.  The FDA continues to promote the use of biomarkers in basic and clinical research, as well as research on potential new biomarkers to use as surrogates in future trials.   Unless otherwise noted, all products are for research use only, not for use in diagnostic procedures. 

Oncology biomarker research is about translating progress made in identifying relevant biomarkers in basic research into cancer therapeutics that potentially benefit patients. Our approach uses mass spectrometry as the discovery engine to discover new oncogenic or tumorgenic biomarkers. Cancer research techniques using mass spectrometry can be used to discover and validate biomarkers that can be used for early detection of cancer, proteomics in cancer prognostics, proteogenomics and monitoring response to therapy.  

HRMS in Clinical Research

From Targeted Quantification to Metabolomics

Dr. Rochat Bertrand, Ph.D., Head of LC-MS facility with CHUV University Hospital in Lausanne, Switzerland discusses the potential advantages high resolution accurate mass spectrometry offer.  He poses a paradigm shift to offer one workflow for doing qualitative and quantitative analysis and how to apply this technology to improve clinical research.

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From Targeted Quantification to Metabolomics

Discovery workflows

Clinical & Translational Research Workflow

Biomarker discovery

Protein biomarker discovery uses a sample cohort to go from 10s of 1000s of proteins in the human proteome to a smaller sample that are differentially expressed in disease and control samples.

Overview for bottom-up proteomics
Bottom-up proteomics serves as the basis for much of the protein research undertaken inmass spectrometry laboratories today. The term ”bottom-up” implies that information about the constituent proteins of a biological sample are reconstructed from individually identified fragment peptides. To facilitate bottom-up MS analysis, proteins are subjected to proteolytic digestion, typically using trypsin. The resulting peptides are usually separated using one or more dimensions of liquid chromatography. The LC eluent is interfaced to a mass spectrometer using electrospray ionization and the fragment peptides are analyzed by mass spectrometry.

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Post translational modification analysis
Nearly all proteins undergo chemical modifications after translation. These post-translational modifications (PTMs) play crucial roles in functional proteomics, regulating the protein structure, activity, and expression. PTMs regulate interaction with cellular molecules such as nucleic acids, lipids and cofactors, as well as other proteins. PTMs can occur at any moment in the "life cycle" of a protein, influencing their biological function in processes such as initiating catalytic activity, governing protein-protein interactions, or causing protein degradation.

Glycosylation and phosphorylation are of particular interest to researchers because they are critical pathways for signaling, activation, and often give insight into disease states. Analysis of PTMs by mass spectrometry using multiple fragmentation techniques yields the most comprehensive structural characterization of modified proteins.

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Relative quantitation
To understand the functions of individual proteins and their place in complex biological systems, it is often necessary to measure changes in protein abundance relative to changes in the state of the system. These measurements have traditionally been performed using Western blot analyses. More recently, modern proteomics has evolved to include a variety of technologies for the routine quantitative analyses of both known and unknown targets.

Discovery-based relative quantification is an analytical approach that allows the scientist to determine relative protein abundance changes across a set of samples simultaneously and without the requirement for prior knowledge of the proteins involved. Here we describe three commonly used techniques for relative quantitation of unknown protein/peptide targets using mass spectrometry.

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Biomarker validation and verification

Targeted quantitation
Demand for targeted quantitation experiments among scientists and clinicians has undergone explosive growth in recent years. The objective of targeted protein quantitation experiments is to determine the protein and/or peptide expression levels of known targets in biological systems. The experiment may be designed to determine either the relative levels of the target species or the absolute levels. The scope of these experiments can range from the analysis of individual samples in a research environment to the assessment of thousands of samples in a clinical research setting.

Mass spectrometry-based targeted quantitation requires a priori knowledge of the molecular targets, as well as of the general properties of the samples in which they are contained. At a minimum, the scientist must know the molecular weight of the targeted species. Knowledge of additional properties of the targets, such as their LC elution times, their expected range of expression levels, and their dynamic range, as well as knowledge of the characteristics of the background matrix, will all help greatly in designing a successful targeted quantitation experiment.

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Top-down proteomics
Complex research endeavors such as the investigation of cell signaling pathways, disease state characterization, and biomarker discovery have all benefited from advances in mass spectrometry.  Such pursuits often employ mass spectrometry-based bottom-up protein identification techniques. These same research endeavors would, in many cases, also benefit from high-throughput top-down mass spectrometric analyses. The main advantages of the top-down approach include the ability to detect degradation products, sequence variants, and combinations of post-translational modifications.

In a Nature publication by Neil Kelleher and co-workers, over one thousand unique gene products were identified from human cells in a single experiment. This accounted for over three thousand differentially modified species. High-resolution mass spectrometry is not only essential to resolve co-eluting intact proteins but also to resolve the isotopic peaks of the highly charged large molecules for charge state determination and accurate mass determination.

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Intact protein characterization

Top-down protein characterization by mass spectrometry is an emerging technology that has several advantages over bottom up sequencing. Top-down analysis initially involves accurate measurement of molecular weight of intact protein followed by the fragmentation of the molecular ion in the gas phase. Top-down analysis facilitates direct observation of C- and N-termini for identification of truncations, preserves the relationship between modifications in any given isoform and allows quantitative differentiation between isoforms.

High mass accuracy and high mass resolution are absolute requirements for this approach due to the complexity of MS/MS data. For more complex intact-protein mixtures, however, on- or off-line LC separation (hyperlink to sample separation section in Translation area) may be required to reduce precursor spectral complexity and minimize ion suppression.

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Featured translational research products

Featured resources

Mass Spectrometry-Based Translational Proteomics

An Enhanced Immunoaffinity Enrichment Method

Using the MS immunoassay–SRM workflow, a standard high-throughput method for developing targeted biomarker identification of proteins in human plasma and serum for clinical research was developed.

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Applications of LC-MS

Development and Research of a Highly Sensitive LC-MS Research Method for Quantification of a Cholesterol Protein in Plasma

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key player in the regulation of circulating low-density lipoprotein cholesterol (LDL-C). Both the distinct forms observed in plasma and posttranslational modifications (PTMs) described in cell-based studies are likely to affect its function and thereby LDL-C levels.

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