On‐line Method for Identification of Native Proteins using pH‐Gradient SAX Chromatography and Reversed Phase Chromatography‐Mass Spectrometry of Tryptic Peptides

A method for identification of proteins based on their pI values and peptide mass fingerprint (PMF) using an on‐line system has been developed. Proteins were separated by pH‐gradient strong anion exchange (SAX) chromatography and trapped on C4 reversed phase (RP) columns. Trapped proteins were on‐line transferred to an immobilized trypsin reactor, and the resulting peptides were subsequently on‐line transferred to an RP analytical column via a combination of RP and weak cation exchange (WCX) trap columns. The tryptic peptides were identified by electrospray ionization time‐of‐flight mass spectrometry (ESI‐TOF‐MS). Three model proteins were used for method development and basic human plasma proteins were subjected to identification. On‐column trypsination is compatible with high acetonitrile concentration. The analysis time was 180 min for the first fraction and an additional 80 min for each subsequent fraction selected for protein identification.