C. E. Costello
This workshop was presented at the 1993 ABRF Annual Meeting and was organized to present a summary of the current state-of-the-art of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for biopolymer analysis and to provide a forum for discussion of experimental details. Panel participants included Catherine E. Costello, MS Resource, Mass. Inst. of Technology, Chair; T. William Hutchens, Dept. of Pediatrics (CNRC), Baylor College of Medicine (present address, Univ. of California, Davis); Matthias Mann, Protein and Peptide Group, European Molecular Biology Laboratory; Henrik Rahpek-Nielsen, Dept. of Molecular Biology, Odense University; John Stults, MS Laboratory, Genentech Inc.; and Martha M. Vestling, Dept. of Chemistry and Biochemistry, University of Maryland, Baltimore County.
Samples are prepared for MALDI mass spectrometry by mixing a small volume of a solution (10^-5 to 10^-7M) with an excess (10^2-10^4-fold) of a matrix material dissolved in the same solvent or solvent mixture; 0.5- 2ul of the resulting solution are applied to a metal target and allowed to dry. A crystalline deposit of the sample/matrix mixture usually forms, since most of the common matrix materials are solids. The matrix is chosen based on its absorption at the wavelength of the laser irradiation to be used and its MALDI compatibility with the compound class to be analyzed. The latter property is determined empirically by comparison of the spectra recorded in various matrices, the analyst being guided by results reported in the literature, experience, rumor and intuition. By now, there have been suitable matrices described for most classes of biomolecules. The introduction of new matrices is frequent: the list of appropriate materials is continuously increased. Some popular UV matrices and their favored applications are shown in Table 1. For IR-MALDI at 2.94 um, succinic acid is most widely useful.
During the MALDI measurement, a portion (usually about 100 um in diameter) of the sample/matrix spot is irradiated with a laser beam whose movement and power are controlled by the operator. Most current systems utilize a small, self-contained nitrogen laser, operating at 337 nm. The use of a video camera to monitor the process has been found very helpful, and several commercial systems now offer this option. Upon irradiation, the energy transfer to the light-absorbent matrix causes excitation and an expulsion of material in the region, resulting in the desorption and ionization of the analyte. The ions thus generated are mass- analyzed and detected. The pattern of their mass-to- charge ratios forms the mass spectrum. For this type of experiment, a linear or reflectron time-of-flight (TOF) mass analyzer is particularly well suited, because of its compatibility with a pulsed ion beam, wide mass range, high transmission and simplicity. Ion traps, double-focusing magnetic instruments and Fourier transform mass spectrometers have also been used for MALDI studies. Papers in the reading list below include descriptions of the various analyzer types.
Figure 1 shows the positive-ion MALDI-TOF mass spectrum of bovine insulin, recorded with sinapinic acid matrix. Abundant singly- and doubly-charged molecular ions, (M+H)^(+) and (M+2H)^(2+) respectively, are present, as well as a signal for a low abundance dimer (2M+H)^(+) formed during the ionization process. The inset shows that the molecular ion region also contains minor species that correspond to (M+Na)^(+) and (M+H+sinapinic acid)+ and its dehydration product. These extraneous peaks can be resolved in this region, but at higher masses, they would contribute to the main peak, shifting its centroid upwards and thus contributing to erroneously higher mass measurements. For accurate mass determination, therefore, it is important to minimize the contribution of such adduct peaks by removal of salts and choice of a matrix that shows minimal adduct formation. Below m/z 20,000 the mass measurement accuracy of MALDI-TOF MS is generally quoted as about +/-0.1% with external calibration and +/-0.01% when internal standards are employed. Here, around m/z 6000, that error would be +/-6 daltons and +/-0.6 daltons respectively.
For larger biopolymers, adduct formation and the presence of broad peaks due to sample microheterogeneity often lower mass assignment accuracy somewhat. However, the determined value remains independent of structural dissimilarities to standards, unlike estimates based on chromatography (including electrophoretic gels) which may be subject to large errors due to differences in the properties that govern sample mobilities. The appearance of numerous multiply-charged peaks (M+nH)^(n+) in the spectra of larger compounds in certain matrices provides additional values which may be averaged to yield improved mass accuracy. Figure 2 shows the spectrum acquired for a non-glycosylated peptide with molecular weight near 45 kDa. When an internal standard was used with this protein, the measured mass was only 15 u different from the value calculated from the gene sequence.
MALDI MS measurements may be carried out by operators with little or no previous experience in mass spectrometry, and the process lends itself to automation for straightforward samples. It is important, even so, that the operator training, sample preparation and data interpretation be performed under the guidance and initial supervision of a person who is aware of the methods for optimization of data acquisition, as well as the possible pitfalls. The manipulation of subnanomole or subpicomole amounts of material (e.g. spots recovered from gels) requires careful attention to minimizing the sources of contamination and contact with sites likely to cause sample loss. The universality of the ionization method means that contaminants will be easily detected, as well as the compounds of interest.
The information content of MALDI MS spectra may be increased by the examination of samples in a variety of matrices, to circumvent problems due to compound class selectivity or size discrimination. Although these are less a factor than in other methods, they still must be considered as potential sources of error. Additional experiments that may shed light on the total components in mixtures and/or detailed structures of individual components include change in polarity (i.e., operation in the negative-ion mode), and multistage mass analysis by recording of the spectra of metastable ions with suitable scans of a reflectron TOF instrument or MS/MS measurements utilizing collision-induced decomposition on sector, FTMS or ion-trap instruments. MALDI metastable and MS/MS methods, while not yet routine, show great promise. Also helpful for structure elucidation are sample modifications such as derivatization (permethylation, peracetylation), or chemical (Edman, CNBr) or enzymatic (proteases, exo- and endo-glycosidases, endo-nucleases) degradation to cause molecular weight shifts that yield sequence information; for small samples, these can all be conducted on microscale levels. Enzyme-catalyzed reactions (e.g., phosphorylation/dephosphorylation) and metal-binding processes can also be followed in situ as conditions are modified. Affinity based approaches can be used advantageously to discriminate in favor of the compound(s) of interest during microscale chromatographic separations or selective binding to specially prepared probe tips.
For samples that are contaminated with salts, buffers or other relatively soluble materials, washing of the sample-laden probe tip may remove the impurities yet leave the sample and matrix largely in place. Proper choice of buffer can improve results, e.g., use of n- octyl glucoside was found to yield spectra covering a much higher mass range for V8 protease digestion products compared to SDS, whose residual presence can hinder the ionization process. Matrix additives such as sucrose may serve multiple purposes: to complex adventitious salts and thus minimize their interference and/or to absorb excess local energy buildup. A sample that has been separated on a gel may be blotted to a membrane and analyzed by MALDI while it remains on the membrane. After initial MALDI mass measurement, the membrane-bound sample may be digested or subjected to Edman degradation and another MALDI mass spectrum recorded to determine the weights of the digestion products. The data obtained in such experiments may be compared to protein sequence databases using desktop software. Recovery of low levels of proteins and peptides from gels is not yet routine; success depends both on the skill of the analyst and the cooperation of the sample. Recent progress in this area has been considerable and further refinements of the technique are likely.
MALDI mass spectrometry is still a very rapidly developing field, but there is already a range of instruments available to suit the needs and technical level of most biochemical and chemical laboratories. Bench-top instruments are convenient and straightforward to operate and to maintain, and the data systems are increasingly user-friendly. Top-of-the-line instruments allow flexibility in experimental design and thus encourage innovation. Most systems may be upgraded as needs and skills grow, and many design improvements can be retrofitted. Thus, today, MALDI may be utilized fairly routinely in the protein laboratory for measurements that are otherwise difficult or impossible. Better yet, it can also be expected to continue its stunning growth into an even more powerful technique in the future.
A Brief Reading List on MALDI MS of Peptides, Proteins
and Other Biopolymers
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