- Shotgun Proteomics For Biomarker Candidate Discovery
- Targeted Quantitative Analyses of Biomarker Candidates
- Antibody Development for Biomarker Candidates
Shotgun Proteomics For Biomarker Candidate Discovery
Shotgun proteome analysis platforms based on multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide a powerful means to discover biomarker candidates in tissue specimens. We digest tissue or biofluid specimens to tryptic peptides and then fractionate peptides by isoelectric focusing (IEF) (Figure 2). The peptides in each IEF fraction are then analyzed by reverse phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The objective of the analysis is to generate a collection of MS/MS spectra from as many of the peptides in the sample as possible. Each MS/MS spectrum encodes the sequence of a peptide; these sequences are determined by searching the spectra against a database of sequences corresponding to all known human proteins. The resulting collection of identified peptide sequences is then assembled into an inventory of proteins that can account for the identified peptides. Because the likelihood of collecting an MS/MS spectrum of a peptide increases with the amount of the peptide in a sample, the numbers of MS/MS spectra that map to each protein provide a preliminary estimate of the amount of each protein in the sample (Figure 3).
Comparisons of the MS/MS datasets for tissue specimens (e.g., normal vs. cancer) thus allows the identification of proteotypes, which are the features of proteomes that distinguish one tissue type from another. The ultimate objective of these shotgun proteomics inventories of normal, precancer and cancer tissues is to identify cancer-related proteotypes. The proteins that comprise these proteotypes are the targets of our further studies to identify biomarkers for the cancers.
Targeted Quantitative Analyses of Biomarker Candidates
The next stage of the biomarker pipeline requires the rapid development of assays to the many biomarker candidate proteins that comprise cancer-specific proteotypes. This requires an analytical technology that can measure the presence of a specific peptide indicative of the presence of the corresponding protein in a tissue or plasma sample. We employ liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) to do targeted measurements of individual proteins (Figure 4). In this approach, we first select one or more peptide sequences from the target protein that are unique to that protein. We then take advantage of prior knowledge of how that peptide fragments in MS/MS to configure a very specific detection method. In short, we set the MS instrument to target only species that have the same mass as the peptide AND also yield fragment that are consistent with that peptide sequence. This approach offers the most selective method available for measuring specific peptides and their corresponding proteins. The greatest challenge in this approach is the detection of a small amount of a peptide in very complex biospecimens (e.g., plasma or tissue samples). We are exploring the combined application of antibodies to target proteins to enrich them from complex samples, followed by targeted LC-MRM-MS analyses.
Antibody Development for Biomarker Candidates
A key objective of the biomarker development pipeline is the development of antibodies against biomarker candidates. These antibodies may include both polyclonal antibodies (generated in rabbits) as well as monoclonal antibodies (generated in mice). Both efforts involve collaborations between the Ayers Institute and the Monoclonal Antibody Core in the Vanderbilt Institute of Chemical Biology. We have developed an antibody development program to generate rabbit polyclonal antibodies to the N-terminal and C-terminal 150 amino acids of sequence for each biomarker candidate of interest. The Ayers Institute has provided funding for personnel and operations costs for this core with the objective of expanding throughput for antibody development. The polyclonal antibodies generated as described above are used to develop hybrid immunoaffinity capture /LC-MRM-MS assays to rapidly screen biomarker candidates (Figure 5). The approach involves capture of the protein biomarker candidate with an immobilized polyclonal antibody, followed by proteolytic digestion and selective detection of the resulting peptides by LC-MRM-MS. Variants of this approach are also being explored, all of which have considerable advantages over conventional ELISAs, which are time-consuming and costly to develop, particularly for multiple biomarker candidates. The development and application of polyclonal antibodies for screening is accompanied by development of monoclonal antibodies in the core, which will be used for ELISAs or similar immunoassays for protein biomarker candidates.