Stabilization of N-terminal cysteines in HPLC-HRMS quality control of peptide pools

Abstract Synthetic peptide pools are used in experimental and clinical immunology, for example, cancer immunotherapy. The analytical characterization is challenging due to the similarity of peptides, usually only a pre-characterization single peptides performed. However, regular quality control mix would be highly desirable. Therefore, high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) method pool was developed. Peptides were synthesized purified > 90% each. lyophilized combined containing 32 analyzed by HPLC-HRMS. Quantification carried out UV detection, identification obtained spectrometry. Before separation on capillary reversed-phase column, alkylation thiols Best results with linear gradient from 96% solvent A (water 0.05% TFA), 4% B (acetonitrile 0.04% TFA) 56% A, 44% 100 minutes. Without sample preparation, N-terminal cysteine will yield almost no signal. This surprising effect could eliminated using iodoacetic acid (IAC) as an reagent. amount IAC reductant (Tris(2-carboxyethyl)phosphine (TCEP)) carefully optimized avoid overalkylation. By combination HPLC HRMS, developed complex synthetic pools. In contrast analysis analyzing requires stabilization cysteine-containing peptides. Good