Investigation of sample preparation and instrumental parameters in the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of noncovalent peptide/peptide complexes.
The application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to the direct detection and investigation of noncovalent solution-phase complexes is far from being routine and some principal problems and questions still exist. Therefore, this study systematically investigates several main problems, namely, the effect of sample preparation and some instrument-related parameters on the stability of the noncovalent complexes as well as the formation of nonspecific cluster ions in the case of the MALDI-MS analysis of specific peptide/peptide complexes. The complexes formed between biologically active fragments of human gastrin I, which contain the sequence motif EEEEE, and different peptides, which contain the interacting sequence motifs RR and RKR, were chosen as examples. A broad variety of MALDI matrices and sample preparation protocols were screened systematically and evaluated. The two 'less acidic' matrices 2,4,6-trihydroxyacetophenone and 6-aza-2-thiothymine, in combination with carefully selected solvents and additives, turned out to allow the reproducible detection of the solution-phase peptide/peptide complexes with good intensity, whereas the classical MALDI matrices could not be applied with the same success. Because both matrices also tend to induce the formation of nonspecific cluster ions, control experiments using nonbinding peptides were performed to definitely prove the specificity of the detected complexes. In contrast to the sensitivity of the peptide/peptide complexes to solution-phase conditions, the gas-phase stability during desorption/ionization was found to be extraordinary high. Neither the application of high laser fluence nor switching from continuous to delayed extraction mode as well as variation of the delay time up to 520 ns had considerable effect on the relative intensities of the specific peptide/peptide complexes.
Zehl
Martin
M
Institute of Chemical Technologies and Analysis, Vienna University of Technology, Getreidemarkt 9/164, A-1060 Vienna, Austria.
Allmaier
Günter
G
D000596:Amino Acids / Q000737:chemistry; D005755:Gastrins / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000596:Amino Acids; D005755:Gastrins; D007202:Indicators and Reagents; D010446:Peptide Fragments; D010455:Peptides