Peptides biomarker identification

FIGURE 1 Proteomics pipeline used for the identification, characterization, and detection of species-specific peptide biomarkers for fish authentication purposes. 

Lopez, M.F., Mikulskis, A., Kuzdzal, S., et al (2007) A novel, high-throughput workflow for discovery and identification of serum carrier protein-bound peptide biomarker candidates in ovarian cancer samples. Clin. Chem., 53 (6), 1067-1074. 

Traditional biochemical techniques such as liquid chromatography (LC), gel electrophoresis, capillary electrophoresis (CE), and mass spectrometry (MS) have been widely used for the complete analysis of salivary proteins and peptides. The recent advances in these technical approaches applied to peptidomics have allowed a better comprehensive analysis of peptides in human whole saliva, envisioning the identification of potential salivary biomarkers of oral and systemic diseases. Sample preparation is a critical experimental step for the successful identification of peptides using MS-based approaches, for their quantitation and identification of PTMs. 

Identification of new biomarkers of OP exposure may also lead to an understanding of why some people are intoxicated by low doses of OP that have no effect on the majority of the population. The new motif for OP binding to tyrosine may lead to new antidotes for OP poisoning for example, peptides containing several tyrosines and several arginines may be effective OP scavengers. 

The combination of MALDI-TOF MS and capillary LC/MS/MS was recently described for the identification of disease state markers in human urine. In this study, urine proteins obtained from emphysema patients were separated on 2-D gels and selected spots were digested with trypsin and analyzed by MALDI-TOF. A database search using Protein Prospector identified a potential biomarker for emphysema as human alpha-1-antitrypsin (AlAT). The corresponding MALDI spectrum contained nine out of 18 peptides with masses that match the expected tryptic digest fragments for AlAT. 

Even though the microfluidic-MS analysis was performed with low injection volumes, it enabled the identification of five putative biomarkers 39,40 proliferating cell nuclear antigen (PCNA), cathepsin D and keratins K8, K18 and K19 (Table 7.1). All corresponding peptides that identified these proteins had p < 0.001, confirming the reliability of the match. PCNA is a protein involved... 

Results and discussion Over 2,000 data components were registered initially using the evaluation software. The data set was reduced with various filters and probability stringency actions to a subset of 14 compounds that could be evaluated as potential markers. Partial identification of the 14 compounds (by MS/MS and accurate mass measurement) showed that 10 were lipidic (molecular mass 240-530 Da), and the others were peptides ( 1 kDa). Notably, the concentrations of all but one of the candidate biomarkers were depressed (1.5- to 5.6-fold) in the pathological samples when compared to controls. Results were consistent in both plasma and serum. 

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