Low-Temperature Mobile Phase for Peptide Trapping at Elevated Separation Temperature Prior to Nano RP-HPLC-MS/MS

Nano HPLC-MS/MS separation and detection of peptides for proteomic analysis is usually performed upon tryptic digest of proteins and peptide pre-concentration on trap columns. Pre-concentration on trap columns is needed for sample wash (removal of salts and impurities), sample focusing prior to separation, and volume reduction. Usually, trap columns are mounted on selection valves close to the separation column in order to keep the void volume low and to enable injection of large sample amounts onto nano-separation column. Since separation columns are operated at elevated temperature of ě45 ̋C and they are mounted on the same valve as the trap column (in the column oven); loading samples at elevated temperature will result with significant loss of analytes. A method for loading samples on a trap column at 60 ̋C was developed and optimized. No sample loss was observed when the optimized method was used for analysis of standards and of complex biological samples.