Capillary Liquid Chromatography/Mass Spectrometry for Peptide and Protein Characterization

Abstract

Developments in capillary column liquid chromatography have been driven in part by the need to develop an effective interface between liquid chromatography and mass spectrometry (LC/ MS). Although the cost of commercial columns and pumping systems capable of gradient elution is relatively high, many aspects of the technology are relatively simple to do in house and easily adapted to existing instrumentation. The vacuum environment of the mass spectrometer can accept only a few microliters per minute of solvent flow. This can be accomplished by splitting higher flows or through the use of a novel pumping system that delivers gradients at low flow rates. The interface to the mass spectrometer is most easily accomplished using electrospray ionization. Electrospray has the advantage of producing multiply charged species that permit the analysis of large protein structures on mass spectrometers with limited mass range. Samples eluting from a capillary column are in an ideal form for electrospray mass spectrometry. Concentrations are high and components of interest have been separated from contaminants such as salts that interfere with the electrospray process. Thus, mass spectral analyses can be obtained on samples that are otherwise difficult or impossible to do. The accuracy of electrospray mass spectrometry is sufficient to monitor small changes in the masses of large proteins. This feature is very useful for studying the interactions of proteins with substrates and inhibitors. IC/MS analysis of peptide mixtures resulting from enzyme digestion of large proteins is very useful for rapidly confirming the sequence of recombinant proteins. When combined with automated methods of obtaining fragment ion spectra on individual peptides during an LC/MS run, it is possible to very quickly determine the location of single amino acid changes in mutant proteins.