Targeted determination of the early stage SCLC specific biomarker pro-gastrin-releasing peptide (ProGRP) at clinical concentration levels in human serum using LC-MS.

Pro-gastrin-releasing peptide (ProGRP) is used as a specific diagnostic marker for small cell lung cancer (SCLC), a rapidly growing neoplasm with high mortality. Our object was to develop an LC-MS method for the detection and quantification of ProGRP in human serum using the specific tryptic digestion product NLLGLIEAK (m/z 485.8 for [M + 2H](2+)). For this purpose the sample pretreatment, clean-up, enrichment, and LC-MS conditions were evaluated. Sample pretreatment was carried out using ACN precipitation to decrease the sample complexity. Although ProGRP (31-98) standards were soluble in 99% ACN, it showed that optimal signal intensities were obtained by adding ACN to the serum in a 1:1 ratio v/v. A simplified tryptic digest protocol was carried out using 100 mM triethanolamine buffer to ensure pH stability during the whole procedure. The simplified protocol also includes omission of reducing and alkylating reagents. Necessary additional sample clean-up was achieved by trapping NLLGLIEAK on a RAM column (ADS-C8) which was back-flushed onto the analytical BioBasic C8 column. Volume of injection, sample enrichment, and column capacity are among the factors optimized to reach a mass LOD of 150 pg on column (OC) ProGRP (31-98). Detection of ProGRP in the serum sample of a patient suffering from SCLC with a clinically relevant concentration shows the potential of the method in diagnosis, prognosis, and monitoring of the disease.