Establishment and application of a high-quality comparative analysis strategy for the discovery and small-scale validation of low-abundance biomarker peptides in serum based on an optimized novel peptide extraction method

摘要

Low-abundance native peptides are an attractive target for the discovery of disease biomarkers. However, validating candidate peptides is difficult due to challenges associated with precise peptide identification and development of high-throughput assays using specific antibodies. Therefore, a highly reproducible and sensitive strategy based on effective peptide enrichment methods is needed to identify clinically useful biomarkers. We optimized our novel differential solubilization (DS) peptide extraction method to selectively enrich peptides less than 6 kDa, using tricine-SDS-PAGE to evaluate the optimization. The modified DS method was combined with liquid chromatography-mass spectrometry (LC-MS) using conventional high-performance liquid chromatography (HPLC). The reproducibility and sensitivity of the proposed strategy were sufficient to enable discovery of low-abundance (ng/mL range) candidate biomarker peptides. A total of 40 serum samples collected pre- and post-surgery from renal cell carcinoma (RCC) patients were analyzed, resulting in discovery of 2 peptides that are upregulated and one peptide that is downregulated in pre-surgery RCC patients. These peptides were validated using 40 serum samples collected pre- and post-surgery from bladder tumor (BT) patients. Two candidate peptides that were upregulated in pre-surgery RCC patients were not upregulated in the sera of the pre-surgery BT patients. Finally, we propose 2 candidate marker peptides that could be used to detect RCC.