Mass spectrometry in peptide and tumor marker analysis

Abstract

Integration of preparative protein and peptide separations with analytical techniques, in particular mass spectrometry (MS), is important. This thesis describes preparation via capillary electrophoresis and two-dimensional (2-D) gel electrophoresis for identification and structure determination of polypeptides. Protocols for protein identification via mass spectrometry were developed. Applications in sequence determination of peptides and in screening of proteins extracted from solid tumor material were tested.

Preparative capillary electrophoresis of peptide samples and subsequent characterization of fractionated material with matrix-assisted laser desorption ionization (MALDI)-MS is feasible provided the same electrolyte is not used for both separation and collection because of a risk for signal suppression in the mass analysis. To avoid suppression, separate electrolytes were used, phosphate buffer for good resolution in the separations and trifluoroacetic acid for improved signal to-noise ratio and resolution of sodium adducts in the mass analysis. To increase the preparative capacity in capillary electrophoresis and to prevent wall adsorption and band-broadening, a low amount of ethylene glycol was used in both sample and buffers.

C-terminal sequence analysis of peptides was carried out using a mixture of two carboxypeptidases, CPP and CPY. The resulting amino acid sequence was determined via MALDI-MS. To distinguish Lys from Gln, which have similar mass, Lys was derivatized to homoarginine. For ladder sequencing of peptides containing Cys, this amino acid was converted to 4-tialaminine.

The protein expression patterns of oncogene-transformed cell lines and tumor cells of different malignancies from breast, lung and ovary were analyzed by 2-D gel electrophoresis. The purpose was to find proteins which can be of potential use as tumor markers. Different protocols were tested for isolation of the protein fragments from the gel matrix. In-gel digestion using modified trypsin with extensive washings to remove salts and detergents was essential, together with precautions to avoid introduction of keratin contaminants and polymers. Both MALDI-MS and ion-trap LC (liquid chromatography)-MS/MS were applied for identification of gel-separated proteins. In general, MALDI-MS is more sensitive than ESI (electrospray ionization)-MS, less affected by contaminants and can serve as a first screening step as several samples can be analyzed sequentially. LC on line with ESI-MS means sample clean-up before analysis of separate peptide components. This set-up is possible to automatize and suppression is reduced, which both are advantages in relation to nanospray technology.