High-performance displacement chromatography-mass spectrometry of tryptic peptides of recombinant human growth...

The combination of high-performance displacement chromatography with continuous flow fast atom bombardment (FAB)-mass spectrometry (MS) offers a means of overcoming the sample capacity limitations imposed by the low flow-rates tolerated in microbore systems employed for directly coupled liquid chromatography-MS. Displacement chromatography is performed at high concentrations with the same equipment and columns as typically used in chromatography at low concentrations. By using this mode of chromatography with a solution of cetyltrimethylammonium bromide as the displacer, the capacity of a reversed-phase column can be increased 50- to 100-fold for separation of a tryptic digest of biosynthetic human growth hormone. Despite the high load, the use of displacement chromatography allowed high-resolution separation of the complex mixture of eighteen major components. On-line analysis by continuous flow FAB-MS yielded high-quality spectra of these peptides and demonstrated that sharp, single-component bands can be obtained in this separation. Along with the major fragments, the chromatogram showed other peptides originating from protein variants in the sample, from non-specific cleavage in the enzymatic digest or from autolysis of trypsin. On-line analysis also allowed selective ion monitoring of the column effluent for individual peptides and confirmed the high efficiency and resolution obtained by preparative displacement separations on HPLC columns and equipment.