2-DIMENSIONAL MICROCOLUMN HPLC COUPLED TO A SINGLE-QUADRUPOLE MASS-SPECTROMETER FOR THE ELUCIDATION OF SEQUENCE TAGS AND PEPTIDE-MAPPING

The system described here uses comprehensive two-dimensional liquid ch romatography to separate the mixture of peptides resulting from the en zymatic digestion of a protein. This system couples size exclusion liq uid chromatography (SEC) to reversed-phase liquid chromatography (RPLC ) to affect first a separation based on size followed by a separation based on hydrophobicity. The microcolumn RPLC allows the use of second -dimension flow rates 20-fold less than previously reported, which con comitantly increases the concentration of the peaks. This second dimen sion of chromatographic separation is directly coupled to an electrosp ray mass spectrometer for the detection of peptides as well as for the determination of their molecular weights. High-resolution peptide map s have been from as little as 15 pmol of digested protein. The high se nsitivity which results from the increased peak concentrations on the reversed-phase microcolumns permits individual peptides to be sequence d using in-source collision-induced dissociation (CID) on this single- quadrupole mass spectrometer. A low orifice voltage in the electrospra y source's interface region is used for intact peptide molecular weigh t determination. A high orifice voltage on a subsequent scan dissociat es the peptide and determines the sequence of about three residues. Kn owing enzyme specificity, peptide molecular weight, and a partial sequ ence allows protein databases to be searched for the protein's identit y with a high level of accuracy. Tryptic digests of bovine serum album in are used to demonstrate the elucidation of sequence tags as well as for traditional peptide mapping experiments. (C) 1998 John Wiley & So ns, Inc.