Sung Hee Ahn

A Simple Method for Quantification of Peptides and Proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

Even though matrix-assisted laser desorption ionization (MALDI) is a powerful technique for mass spectrometry of peptides and proteins, it is not quite useful for their quantification that is one of the outstanding problems in quantitative proteomics. The main difficulty lies in the poor reproducibility of MALDI spectra. In this work, a simple method to circumvent this problem has been developed. The method is based on a previous observation that the reaction quotient for the matrix-to-peptide proton transfer evaluated in temperature-selected MALDI was nearly constant regardless of the peptide concentration in the solid sample. This implied a direct proportionality between the relative abundance of an analyte ion in a temperature-selected MALDI spectrum and the concentration of the corresponding neutral in the solid sample. This relation has been confirmed by calibration curves obtained for some peptides. Another characteristic of the relation is that it holds even when other analytes are present. This has been demonstrated for mixtures containing peptides and proteins. This and the fact that the method does not require the addition of internal standards allow rapid and inexpensive quantification of any analyte amenable to MALDI.

Quantitative reproducibility of mass spectra in matrix-assisted laser desorption ionization and unraveling of the mechanism for gas-phase peptide ion formation.

In a previous study on matrix-assisted laser desorption ionization (MALDI) of peptides using α-cyano-4-hydroxycinnamic acid (CHCA) as a matrix, we found that the patterns of single-shot spectra obtained under different experimental conditions became similar upon temperature selection. In this paper, we report that absolute ion abundances are also similar in temperature-selected MALDI spectra, even when laser fluence is varied. The result that has been obtained using CHCA and 2,5-dihydroxybenzoic acid as matrices is in disagreement with the hypothesis of laser-induced ionization of matrix as the mechanism for primary ion formation in MALDI. We also report that the total number of ions in such a spectrum is unaffected by the identity, concentration and number of analytes, i.e. it is the same as that in the spectrum of pure matrix. We propose that the generation of gas-phase ions in MALDI can be explained in terms of two thermal reactions, i.e. the autoprotolysis of matrix molecules and the matrix-to-analyte proton transfer, both of which are in quasi-equilibrium in the early matrix plume.

Efficient methods to generate reproducible mass spectra in matrix-assisted laser desorption ionization of peptides.

AbstractIn our previous matrix-assisted laser desorption ionization (MALDI) studies of peptides, we found that their mass spectra were virtually determined by the effective temperature in the early matrix plume, Tearly, when samples were rather homogeneous. This empirical rule allowed acquisition of quantitatively reproducible spectra. A difficulty in utilizing this rule was the complicated spectral treatment needed to get Tearly. In this work, we found another empirical rule that the total number of particles hitting the detector, or TIC, was a good measure of the spectral temperature and, hence, selection of spectra with the same TIC resulted in reproducible spectra. We also succeeded in obtaining reproducible spectra throughout a measurement by controlling TIC near a preset value through feedback adjustment of laser pulse energy. Both TIC selection and 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 α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid as matrix. Based on the utilization of acquired data, TIC control was more efficient than TIC selection by an order of magnitude. Both techniques produced calibration curves with excellent linearity, suggesting their utility in quantification of peptides. Figureᅟ

Matrix Suppression as a Guideline for Reliable Quantification of Peptides by Matrix-Assisted Laser Desorption Ionization

We propose to divide matrix suppression in matrix-assisted laser desorption ionization 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%. 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%. The theoretical basis for the quantification method using a peptide as an internal standard is presented together with its weaknesses. A systematic method to improve quantification of high concentration analytes has also been developed.

Investigations of Some Liquid Matrixes for Analyte Quantification by MALDI

AbstractSample inhomogeneity is one of the obstacles preventing the generation of reproducible mass spectra by MALDI and to their use for the purpose of analyte quantification. As a potential solution to this problem, we investigated MALDI with some liquid matrixes prepared by nonstoichiometric mixing of acids and bases. Out of 27 combinations of acids and bases, liquid matrixes could be produced from seven. When the overall spectral features were considered, two liquid matrixes using α-cyano-4-hydroxycinnamic acid as the acid and 3-aminoquinoline and N,N-diethylaniline as bases were the best choices. In our previous study of MALDI with solid matrixes, we found that three requirements had to be met for the generation of reproducible spectra and for analyte quantification: (1) controlling the temperature by fixing the total ion count, (2) plotting the analyte-to-matrix ion ratio versus the analyte concentration as the calibration curve, and (3) keeping the matrix suppression below a critical value. We found that the same requirements had to be met in MALDI with liquid matrixes as well. In particular, although the liquid matrixes tested here were homogeneous, they failed to display spot-to-spot spectral reproducibility unless the first requirement above was met. We also found that analyte-derived ions could not be produced efficiently by MALDI with the above liquid matrixes unless the analyte was sufficiently basic. In this sense, MALDI processes with solid and liquid matrixes should be regarded as complementary techniques rather than as competing ones. Graphical Abstractᅟ

Spectral Reproducibility and Quantification of Peptides in MALDI of Samples Prepared by Micro-Spotting

AbstractPreviously, we reported that MALDI spectra of peptides became reproducible when temperature was kept constant. Linear calibration curves derived from such spectral data could be used for quantification. Homogeneity of samples was one of the requirements. Among the three popular matrices used in peptide MALDI [i.e., α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and sinapinic acid (SA)], homogeneous samples could be prepared by conventional means only for CHCA. In this work, we showed that sample preparation by micro-spotting improved the homogeneity for all three cases. Figureᅟ

Acquisition of the depth profiles and reproducible mass spectra in matrix‐assisted laser desorption/ionization of inhomogeneous samples

RATIONALE In our previous analysis of the matrix-assisted laser desorption/ionization (MALDI) spectra of peptides, we treated their depth profiles in solid samples as homogeneous. Here, we wanted to determine if the reproducible MALDI spectra and linear calibration curves reported previously would be obtained even when the depth profiles were inhomogeneous. METHODS We derived a formula relating shot-number-dependent ion abundance data in temperature-controlled MALDI with the analyte depth profile in a solid sample. We prepared samples containing peptides, amino acids, and serotonin in α-cyano-4-hydroxycinnamic acid matrix by vacuum-drying and micro-spotting methods, recorded their MALDI spectra, and analyzed them with the aforementioned formula. RESULTS For the samples prepared by vacuum-drying, the analyte depth profiles were inhomogeneous and maximized at the sample surface. Although the MALDI spectra changed as the shot continued, their sum over the entire set of spectra acquired from a spot was reproducible. Similarly, a high-quality calibration curve could be obtained with the spectral data summed over the entire set. Depth profiles were homogeneous for samples prepared by micro-spotting. CONCLUSIONS A method has been developed to obtain a reproducible MALDI spectrum and a linear calibration curve for an analyte with an inhomogeneous depth profile in a solid sample.

Quantification of Carbohydrates and Related Materials Using Sodium Ion Adducts Produced by Matrix-Assisted Laser Desorption Ionization

AbstractThe utility of sodium ion adducts produced by matrix-assisted laser desorption ionization for the quantification of analytes with multiple oxygen atoms was evaluated. Uses of homogeneous solid samples and temperature control allowed the acquisition of reproducible spectra. The method resulted in a direct proportionality between the ion abundance ratio I([A + Na]+)/I([M + Na]+) and the analyte concentration, which could be used as a calibration curve. This was demonstrated for carbohydrates, glycans, and polyether diols with dynamic range exceeding three orders of magnitude. Graphical Abstractᅟ

Quick quantification of proteins by MALDI

Previously, we reported that the matrix-assisted laser desorption ionization spectrum of a peptide became reproducible when an effective temperature was held constant. Using a calibration curve drawn by plotting the peptide-to-matrix ion abundance ratio versus the peptide concentration in a solid sample, a peptide could be quantified without the use of any internal standard. In this work, 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.

Gain Switching for a Detection System to Accommodate a Newly Developed MALDI-Based Quantification Method

AbstractIn matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), matrix-derived ions are routinely deflected away to avoid problems with ion detection. This, however, limits the use of a quantification method that utilizes the analyte-to-matrix ion abundance ratio. In this work, we will show that it is possible to measure this ratio by a minor instrumental modification of a simple form of MALDI-TOF. This involves detector gain switching. Graphical Abstractᅟ