Generation of reproducible MALDI spectra and its application to analyte quantification

Abstract

Matrix-assisted laser desorption ionization (MALDI) is a useful ionization technique for the mass spectrometry of biomolecules. One of the requirements for generating reproducible MALDI spectra is to prepare samples with good homogeneity. Until now, we have not been successful in producing homogeneous solid samples from popular matrixes apart from α-cyano-4-hydroxycinnamic acid (CHCA). In chapter 1, we showed the production of an outwardly homogeneous solid sample by loading a methanol solution of a matrix into a shallow reservoir on a coated MALDI sample plate and then vacuum-drying it. Out of ten popular matrixes tested, seven yielded homogeneous samples. These were 9-aminoacridine, 6-aza-2-thiothymine, CHCA, 2,5-dihydroxybenzoic acid (DHB), ferulic acid, sinapinic acid, and 2,4,6-trihydroxyacetophenone. For MALDI with these matrixes, linear calibration curves plotted in the form of I(A+H+)/I(M+H+) versus analyte concentration were acquired. Features of these matrixes in various aspects of analyte quantification have been examined. In our previous MALDI studies of peptides, we also found that their mass spectra were practically determined by the effective temperature in the early matrix plume, Tearly, when samples were homogeneous. But calculating Tearly was complicated. In chapter 2, we explained another empirical rule that the total number of particles hitting the detector (TIC) was a good measure of the spectral temperature. We also succeeded in generating reproducible spectra throughout a measurement by controlling TIC near a preset value through feedback adjustment of laser pulse energy. TIC control substantially reduced the shot-to-shot spectral variation in a spot, spot-to-spot variation in a sample, and even sample-to-sample variation in MALDI using CHCA or DHB as matrix. This technique produced calibration curves with excellent linearity, suggesting their utility in quantification of peptides. In chapter 3, we proposed to divide matrix suppression in MALDI into two parts, normal and anomalous. In quantification of peptides, the normal effect can be accounted for by constructing the calibration curve in the form of peptide-to-matrix ion abundance ratio versus concentration. The anomalous effect forbids reliable quantification and is noticeable when matrix suppression is larger than 70% for CHCA matrix. With this 70% rule, matrix suppression becomes a guideline for reliable quantification, rather than a nuisance. A peptide in a complex mixture can be quantified even in the presence of large amounts of contaminants, as long as matrix suppression is below 70%. Lastly, we attempted to quantify various molecules. In chapter 4, we quantified proteins by quantifying their tryptic peptides with the aforementioned method. We modified the digestion process

e.g. disulfide bonds were not cleaved, so that hardly any reagent other than trypsin remained after the digestion process. This allowed the preparation of a sample by the direct mixing of a digestion mixture with a matrix solution. We also observed that the efficiency of the matrix-to-peptide proton transfer, as measured by its reaction quotient was similar for peptides with arginine at the C-terminus. With the reaction quotient averaged over many such peptides, we could rapidly quantify proteins. Most importantly, no peptide standard, not to mention its isotopically labeled analog, was needed in this method. In chapter 5, the utility of sodium ion adducts produced by MALDI for the quantification of analytes with multiple oxygen atoms was evaluated. The method resulted in a direct proportionality between the ion abundance ratio I([A + Na]+)/I([M + Na]+) versus analyte concentration, which could be used as a calibration curve. This was showed for carbohydrates, glycans, and polyether diols with dynamic range exceeding three orders of magnitude.