Analyte Quantitation by MALDI

Another problem concerns the suppression of ion formation from an analyte caused by another analyte or by the presence of another constituent (e.g., buffer) in the solution. Since the MS response significantly depends on solvent and sample composition, ion signal intensities of a given analyte do not necessarily correlate with its concentration in the sample. For the quantitative analysis of an analyte of interest by ESI-MS (the same is true for MALDI-MS), the use of an adequate internal standard is therefore mandatory. 

Mass spectrometry is a powerful qualitative and quantitative analytical tool that is used to assess the molecular mass and primary amino acid sequence of peptides and proteins. Technical advancements in mass spectrometry have resulted in the development of matrix-assisted laser desorption/ion-ization (MALDI) and electrospray ionization techniques that allow sequencing and mass determination of picomole quantities of proteins with masses greater than 100kDa (see Chapter 7). A time-of flight mass spectrometer is used to detect the small quantities of ions that are produced by MALDI. In this type of spectrometer, ions are accelerated in an electrical field and allowed to drift to a detector. The mass of the ion is calculated from the time it takes to reach the detector. To measure the masses of proteins in a mixture or to produce a peptide map of a proteolytic digest, from 0.5 to 2.0 p.L of sample is dried on the tip of tlie sample probe, which is then introduced into tire spectrometer for analysis. With this technique, proteins located on the surfaces of cells are selectively ionized and analyzed. 

AU ionization techniques used for quantitative analysis present the possibility of ionization suppression (and sometimes enhancement) of the analyte(s) by co-eluting compounds arising from the sample matrix or elsewhere this phenomenon is a form of matrix effect. This problem is much more serious for API techniques than the others presented in this chapter, with ESI presenting the highest occurrence of matrix effects. Matrix effects are also of concern in MALDI analyses, especially in the quantitation of small molecules. It is always advisable to be aware of the importance of matrix effects in any proposed analytical method and to minimize them as far as possible. Use of an appropriate isotope-labeled standard in conjunction with matrix matched calibrant standards should give reliable results if all other appropriate precautions are taken, but it is important to investigate the possibility of relative matrix effects if one suspects that the matrix in the analytical sample might differ appreciably from that used to make the matrix matched cahbrants. 

Combined SEC-MALDI. Mark-Houwinkr-Sakurada (MHS) Parameters As mentioned earlier, combination of the quantitatively reliable LC techniques with the identification power of MS was used to overcome the limitations inherent in the use of either LC or MS alone. In addition to ESI-MS/SEC, combined applications already mentioned, several approaches to SEC/ MALDI time-of-flight (TOF) MS coupling have also been reported [78-83]. In the case of polydisperse polymers, determination of molecular mass by SEC/MALDI-TOF involves the fractionation of the samples through an analytical SEC. Selected fractions are then analyzed by MALDI-TOF and the mass spectra of these nearly monodisperse samples allow the determination of the MWD moments. Montaudo et al. [81] have tested the reliability of the method for different polydisperse samples, such as poly(methylmethacrylate) (PMMA), poly(dimethylsiloxane) (PDMS), and copolyesters. 

In direct insertion techniques, reproducibility is the main obstacle in developing a reliable analytical technique. One of the many variables to take into account is sample shape. A compact sample with minimal surface area is ideal [64]. Direct mass-spectrometric characterisation in the direct insertion probe is not very quantitative, and, even under optimised conditions, mass discrimination in the analysis of polydisperse polymers and specific oligomer discrimination may occur. For nonvolatile additives that do not evaporate up to 350 °C, direct quantitative analysis by thermal desorption is not possible (e.g. Hostanox 03, MW 794). Good quantitation is also prevented by contamination of the ion source by pyrolysis products of the polymeric matrix. For polymer-based calibration standards, the homogeneity of the samples is of great importance. Hyphenated techniques such as LC-ESI-ToFMS and LC-MALDI-ToFMS have been developed for polymer analyses in which the reliable quantitative features of LC are combined with the identification power and structure analysis of MS. 

Both absolute quantitation and relative quantitation of species in mixtures is of interest in some circumstances. Quantitation in a 5-minute analysis can be achieved by addition of an internal standard, ideally the target microorganism grown in special media to incorporate heavy isotopes92-95 and determination of the relative peak heights of pairs of proteins from the analyte and the standard. Isotope-labeled proteins or peptides, selected to match proteins or peptides characteristic of target microorganisms, can also serve as internal standards for isotope ratio measurement. The addition of unmatched proteins or peptides is less reliable for either ESI or MALDI measurements because of unpredictable suppression in the variable mixture. 

The use of MALDI-MS for the measurement of low molecular mass compounds is widely accepted now [61], but quantification remains problematic. The main problem is the inhomogeneous distribution of the analytes within the matrix [62]. This leads to different amounts of ions and therefore to different signal intensities at various locations of a sample spot. The simplest and most effective way to overcome this problem is the use of an appropriate internal standard [63]. The use of deuterated compounds with a high molecular similarity to the analyte as internal standards leads to a linear correlation between relative signal intensities and relative amount of the compound to be quantified (Fig. 4b) [64]. Using this approach it is possible to quantitate substrates and products of enzyme catalyzed reactions. Two examples were shown recently by Kang and coworkers [64, 65]. The first was a lipase catalyzed reaction which produces 2-methoxy-N-[(lR)-l-phenylethyl]-acetamide (MET) using rac-a-... 

Presently, FAB-MS spectra are routinely used to characterize synthetic tyrosine O-sulfate peptides.152,57,63-671 Since partial hydrolysis of the sulfate ester occurs in the gas phase, quantification of the tyrosine O-sulfate residue by mass spectrometry is not possible, but combined with one-peak assignment in HPLC, FAB-MS represents a powerful analytical tool. On the other hand, partial hydrolysis in the gas phase excludes the presence of sul-fonated species which should be perfectly stable. In early studies the presence of such species were excluded by quantitative recovery of tyrosine upon acid hydrolysis or upon hydrolysis with arylsulfatase.1361 Recently, even MALDI-TOF-MS spectra of CCK-peptides1441 and of conotoxins a-PnIA and a-PnlB 138 were reported which show that in the positive-ion mode the [M + H-S03]+ ions represent the base peaks, while in the negative-ion mode, [M-H]-ions consistently correspond to the base peaks. In the CCK peptides intramolecular salt bridging of the sulfate hemi-ester with proximal positive charges of arginine or lysine side chains was found to reduce the extent of hydrolysis in the gas phase significantly.144,1491... 

Signal intensities in MALDI MS not only depend on the amount of the analyte but also on its chemical composition, for example, the sequence of amino acids in the case of peptides, and may underlie suppression by other components present in the sample [51,52]. Further, the hot-spot formation described above leads frequently to poor shot-to-shot and spot-to-shot reproducibilities. Both factors hamper the use of MALDI MS for quantitative... 

To achieve the best performance in protein identification or quantitation, the extent of protein or peptide separation should match the capabilities of the technique applied in the identification or quantitation step. With LC-MALDI MS and MS/MS as analysis technique, the number of good-quality MS/MS spectra, which can be acquired from one sample spot (LC-fraction) represents one limitation the number of components in one fraction should therefore not exceed this maximum. By increasing matrix concentration more laser shots could be acquired from one spot, but analyte concentration in the crystals and detection sensitivity would... 

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