Report
MALDI MS and Strategies for Protein Analysis
M
atrix-assisted laser desorption/ MALDI, combined ionization (MALDI) ionizes molecules with molecular with enzymatic masses of 100-1,000,000 Da for analysis by MS and provides high sensitivity, high reactions and protein throughput, and simplicity of operation. MALDI was developed based on earller chemistry, provides successes in applying laser desorption to bioorganic molecules and the significant information on improvements in plasma desorption and fast-atom bombardment (FAB) that matrimolecular masses, ces provided. Ten years ago ,he nearly demonstrations in Germany peptide maps, and simultaneous (2) and Japan (2) of the desorption of proions in excess of 60 000 Da initiprimary structure. teinaceous ated worldwide interest and activity and also stimulated the establishment of numerous start-up companies to sunnly the neressary instrumentation This reoort hiehlights how MAL DI MS is beinc used to analv/p proteins and npntides Catherine Fenselau University of Maryland—Baltimore County S0003-2700(97)09033-1 CCC: $14.00 © 1997 American Chemical Society
The matrix in MALDI appears to fill several roles: It absorbs energy from the pulsed laser beam, isolates sample mole-
cules, and provides photoexcited acid or basic sites for ionization of sample molecules in ion/molecule collisions (3). Although the details of energy conversion and sample desorption and ionization continue to be studied, a general understanding of the mechanism is shown in Figure 1 (4). Energy from the laser beam is absorbed by the chromophoric matrix, which rapidly expands into the gas phase, entraining analyte molecules as well. Ionization occurs by proton transfer between excited matrix molecules and analyte molecules presumably in the solid phase and also by collisions in the expanding plume Ions are steered into the mass analyzer which measures m/z usually plotted against ion abundance (Figure 2) Experimental variables in MALDI MS include the matrix and the ratio of matrix to analyte; laser power, wavelength, and pulse width; and the choice of recording either positive or negative ions. Nitrogen lasers with illumination in the UV range
Analytical Chemistry News & Features, November 1, 1997 6 6 1 A
Report
Figure 1 . Schematic mechanism for MALDI using a UV laser. (a) Absorption of UV radiation by the matrix and ionization of the matrix, (b) Dissociation of the matrix, phase change to supercompressed gas, and transfer of charges to analyte molecules. (c) Expansion of matrix at supersonic velocity, entrainment of analyte in expanding matrix plume, and transfer of charges to analyte molecules. (Adapted from Ref. 4.)
(337 nm) are most commonly available on commercial MALDI mass spectrometers, pulsed so as not to pyrolyze the sample. UV-absorbing aromatic compounds are most commonly used as matrices (5), and various aromatic acids provide excellent sensitivity for forming protonated ions. As with FAB (6) and electrospray ionization (ESI) (7), differential desorption is often observed among the components of mixtures analyzed by MALDI MS. Proteins and peptides are frequently analyzed in mixtures, such as mixtures of peptides produced by the cleavage of proteins with the enzyme trypsin. The problem with 662 A
differential desorption in this class of compounds is illustrated in Figure 2, which presents spectra of an equimolar mixture of four small proteins using two different matrices (8). Relative peak heights vary significantly between the two spectra, and differential desorption efficiencies are obvious within each spectrum. Different amounts of fragmentation are also observed with different matrices, such as the loss of sialic acid from glycopeptides (9), phosphate from phosphopeptides (10), and side-chain protecting groups from synthetic peptides (11). Many types of mass spectrometers are used with MALDI, including time-of-fllght (TOF), Fourier transform (FT), Paul trap, magnetic sector, sector-TOF, TOF-trap, and TOF-TOF instruments. These instruments are currentiy supplied by more than a dozen manufacturers. FT analyzers are noted for providing high-resolution analyses of ions desorbed by MALDI, and this combination is becoming increasingly popular. However, TOF analyzers are most commonly used with MALDI. Such ion analyzers can be pulsed synchronously with the pulsed production of ions bv the laser thereby optimizing sensitivity which is currently in the attomole to femtomole range TOF analyzers can transmit an unlimited mass range are ruewd and simnle can be built at low cost In MAL DI provided much of the impetus to develop TOF mass spectrometers (4) The mass accuracy of MAL DI-TOF instniments is generally 0.1-0.01%. and resolution j 1
.•
rapid optimization of tryptophan-specific proteolysis by 3-bromo-2-(o-nitrophenylsulfenyl) skatole (15). MALDI has been used most successfully for analyzing proteins and peptides. It has also been applied to a large variety of drugs, metabolites, and other low-molecular-weight compounds. The mass ranges of susceptible oligosaccharides and oligonucleotides have thus far been more limited; however, because of its speed and sensitivity, MALDI lends itself well lo incorporation with enzymatic and duplexing strategies to characterize these biopolymers. Soluble chemical polymers can also be analyzed and methods development is underway for insoluble chemical polymers MALDI is more sensitive than two other popular condensed-phase ionization techniques, FAB (6) and ESI (7). MALDI produces ions from molecules 100-fold heavier than FAB, and it is more suitable for mixture analysis than ESI because the latter generates ions with many different charge states from each heavy component. The current requirement for a solid sample distinguishes MALDI from FAB and ESI, and has impeded chromato-
on Ann
can exceed 1 part in 20,000. A low-cost instrument that can rapidly determine molecular masses of biopolymers with 0.01% accuracy is a very powerful tool for biochemists, molecular biologists, and the biotechnology industry. MALDI measurements have contributed to many important studies in these areas during the past decade. Examples of applications from hundreds of papers in the literature include characterizing polyglycylation as a post-translational modification of tubulin in axonemal microtubules (12) determining loading values for drugs conjugated to human albumin for raising antibodies or testing new methods for drug delivery (13) desorbing neuropeptides from single neurons in studies of peptide processing characterizing reaction products for
Analytical Chemistry News & Features, November 1, 1997
Figure 2. MALDI spectra of an equimolar mixture of lysozyme (lys), cytochrome c (cyt), ubiquitin (ubi), and aprotinin (apr) in (a) a-cyano-4-hydroxycinnamic acid, 2500:1 (matrix:analyte) and (b) sinapinic acid, 5 0 0 0 : 1 . Laser wavelength is 337 nm. (Adapted from Ref. 8.)
cial techniques must be used to detect the products of slow fragmentation of larger ions such as peptides. Pulsed extraction of ions from the source (time-lag focusing) and fully focused reflectrons in the ion Protein and peptide structures analyzer have been incorporated into TOF In addition to molecular masses, MS has mass spectrometers (4,19) to facilitate historically provided fragment ions, from detection of product ions from, respecwhich information can be deduced about tine structures of peptides and other organic tively, in-source decay and postsource decay. Ion trapping in FT (20) or Paul trap molecules. Under conventional conditions (as in Figure 2), MALDI spectra of peptides (21) instruments also allows ffagment ions to be formed and detected. and proteins do not contain abundant fragment ions. This condition can be an asset if Post-translational and chemical modifimolecular masses are sought However, cations can be characterized by combining MALDI can be combined with enzymatic MALDI and residue-specific enzymatic rereactions and protein chemistry to provide actions. Comparison of peptide maps before structural information at three levels moand after treatment with alkaline phoslecular peptide maps and primary phatase permits the recognition of phosstructure (including sequences and postphorylated peptides. Interdigitation of translational modifications) Some of these MALDI measurements with residuestrategies are presented in Figure 3 The specific glycosidases identifies which pepuse of MALDI to man oeptide mixtures tides are glycosylated and also provides or other proteolytic some sequence information for the oligoenzymes for quality control in the producsaccharide side chains. Although mass tion of a recombinant subtilisin variant is spectra generally provide achiral structural graphic interfacing; however, the solid sample capability has facilitated automation of multiwell sample holders.
information, most glycosidic and proteolytic enzymes have chiral specificity. MALDI MS can be used in protected reaction strategies to provide rapid and sensitive analyses of the chemical and enzymatic modifications allowed at various sites in folded proteins and in noncovalent complexes. Thus, MALDI provides information on the folded structure or on interactions in the complex. Interfacing biochemical separation techniques
Thus far, MALDI has been applied to samples in the solid phase and has not been a candidate for direct interfacing with HPLC or CE. However, ,i tends iiself well to affinitybased separations and gel electrophoresis—sample purification techniques widely used by biochemists and molecular biologists. Both binding partners can be observed when immunoprecipitates are analyzed. Immuno- and affinity chromatography are also effectively interfaced off-line with MALDI. Interestingly, affinity corn-
illustrated in Fimrre 4a (16) Anv hatch to-
batch changes in the primary structure of the protein product can be readily detected in point-to-point comparisons of the peptide mixture generated by incubation with the proteolytic enzyme trypsin. Peptide sequencing by MS is usually based on detecting mass differences associated with various amino acids in the polymer chain. As suggested in Figure 3, carboxypeptidases and aminopeptidases can be used to produce peptide ladders for rapid analysis by MALDI. Laserdesorbed peptide ions can also be subjected to collisional activation to produce fragment ions that reveal sequence information. In a third approach, partially terminated synthesis has also been bined with MALDI-TOF to provide structures of synthetic peptides Figure 4b presents the spectrum of such a peptide mixture or ladder isolated from a single resin bead and the sernience of the major combinatorial product on that bead (17) Such are often used in combinatorial synthesis like other ionization techniques, MALDI produces some analyte ions with sufficient internal energy to decompose. However, the rates and extent are inversely proportional to the number of bonds in the molecular ion (18), and spe-
Figure 3. Strategies for characterizing proteins by MALDI MS and biochemical procedures. Analytical Chemistry News & Features, November 1, 1997 6 6 3 A
Report plexes with one partner immobilized (e.g., on beaded agarose) can be used intact for MALDI. The complex is broken by an acidic matrix solution, the nonimmobilized partner is readily desorbed and analyzed, and the bead or surface-bound partner can be recovered. Strategies combining gel electrophoresis and laser desorption are under development worldwide, with the perception that
MS provides structural information to complement the chemical information implicit in electrophoretic mobility. Comparable amounts of sample are required, and laser desorption is compatible with the 2-D geometry of gel electrophoresis. Desorption of the sample has been demonstrated from electroblotted membranes, directly from plastic-backed gels, and following extraction of individual bands or spots. Chemical
and enzymatic reactions can be carried out in situ to provide peptide maps, and some of these peptides can be partially sequenced by MS techniques to provide data for computer programs that use molecular weights, proteolytic maps, and limited sequences to search libraries of protein and gene sequences. These programs are readily accessible at various e-mail addresses (22) and—in combination with MS—provide a powerful, rapid approach to characterizing protein spots in electropherograms. In one recent example, about 25% of the proteome from yeast strain S288C (whose genome has been sequenced) was separated by 2-D gel electrophoresis (Figure 5). About 80% of the protein spots were characterized by MS measurements combined with database searching (23). However, one of the authors cautions that the approach is still far from routine (24).
What's next? Efforts are underway to improve sensitivity, primarily by implementing and automating microsampling techniques. A striking improvement in sensitivity was demonstrated with a 100 um x 100 um picovial etched in a silica sheet, to which sample and matrix were added under a microscope. A molecular ion was recorded from 2.5 attomoles of bradykinin (25). The reliable analysis of samples in similar wells in 10 x 10 arrays has recently been demonstrated using automated sampling in the x y plane (£6) For this study a matrix solution of 3-hydroxypicolinic acid and 8 fmol of a 25-base nucleotide were added to each well automatically with a piezoelectric pipet Hirfi-quality MALDI spectra were recorded from all 1000 wells in the arrav demonstrating that this automated annroach is sensitive reproducible and reliable The search for improved matrices continues, particularly for use with IR lasers, which potentially offer improved measurements of analytes that are themselves UV chromophores. Liquid matrices are being sought for characterizing noncovalent complexes and reaction monitoring in situ. Figure 4. Peptide mapping and sequencing by MALDI. (a) Comparison of tryptic peptide maps from two production batches of a recombinant subtilisin variant. T4-14 indicates the order in the protein of each peptide released by trypsin. (Adapted with permission from Ref. 16.) (b) Mass spectrum of a 5% portion of the peptide products isolated from a single resin bead. This ladder mixture establishes that the major product has the sequence FQPHH or phenylalanyl glutaminyl prolyl histidyl histidine. (Adapted with permission from Ref. 17.) 664 A
Analytical Chemistry News & Features, November 1, 1997
In another trend, newly available lasers—smaller and less complex—are being combined with smaller analyzers and pumps to provide shoe-box-sized portable detectors for on-site analysis. Soon the laptop computer will be the largest module in the MALDI instrument!
Something New Has Been Added to H Y D R A N A L®
REAGENTS • Better Price • Better Service • Sigma-Aldrich Sigma-Aldrich is now your source for HYDRANAL® Karl Fischer Reagents. Get the same patented References
M. P.; Keough, T. Rapii Commun. Mass Spectrom. .194, 8, 77-81. Demirev, P.; Olthofi, I. K; Fenselau, C; Cotter, R J. Anal. Chem. 1987,59,1951-54. Cotter, R J. Anal Chem. 1992,64, 1027-39 A Marshall, A G.; Grosshans, P. B. Anal. Chem. 1991, 63,215 A-29 A McLuckey, S. A; Van Berkel, G. J.; Goo eringer, D. E.; Gllsh, G. L Anall Chem. 1994, 66,689 A-96 A Sites for protein and peptide searches: http://www.public.iastate.edu/~pedro/ rt_l.html; http://rafael.ucsf.edu/MS-Fithtml; http://expasy.hcuge.ch/ch2d/ pi_tool.html; http://chaitsgi.rockefeller.edu/cgi-bin/ prot-id; http://cbrgiinf.ethz.ch/subsection3_l_3. html; http://www.mann.embl-heidelberg.de/ Services/Peptide Search/Peptide Search Intro.html Shevchenko, A et al. Proc. Nat. Acad. Sci. 1996, 93,14440-45. Mann, M. High Sensitivity MS/MS Sequencing ABRF Newslist, 97-71-06 (ABRF@aecom. yn.edu). Jespersen, S. et al./. Rapid Commun. Mass Spectrom. 1994,4, 581-84. Little, D. P.; Cornish, T. J.; O'Donnell, M. J.; Braun, A; Cotter. R. ..; Koster, H., Anal. Chem. in press.
(1) Karas, M.; Hillenkamp, F. Anall Chem. (18) 1988, 60, 2299-2301. (2) Tanaka, K; Waki, H.; Ido, Y.; Akita, S.; (19) Yoshida, Y.; Yoshida, T. Rapid dommun. Mass Spectrom. 1988,8,151-53. (20) (3) Hillenkamp, F.; Karas, M.; Beavis, R C; Chait, B. T. Anal. Chem. 1991, 63,1193 (21) A-1202 A. (4) Cotter, R. J. Time-of-Flight Mass Spectrometry: :nstrumentation ond Applications io (22) Biological Research; American Chemical Society: Washington, DC, 1997. (5) Fitzgerald, M. C; Parr, G. R; Smith, L. Anal. Chem. .193, 65, 3204-11. (6) Fenselau, C; Cotter, R J. Chem. Rev. 1987,57, 501-12. (7) Gaskel, S.J. Mass Spectrom. 1997,32, 677-88. (8) Vestling, M. M. In Time-of-Flight Mass Spectrometry; Cotter, R J., Ed.; American Chemical Society Symposium Series 549: Washington, DC, 1994; pp. 211-24. (9) Papac, D. I.; Wong, A; Jones, AJ.S. Anal. (23) Chem. 1996, 68, ,215-23. (10) Annan, R S.; Carr, S. A. Anall Chem. (24) 1996,68,3413-21. (11) Schmidt, M.; Krause, E.; Beyermann, M.; Bienert, M. Pept. Res. 1195,8, 238- (25) 42. (12) Redeker, V. et al. Sciencce199,266, (26) 1688-91. (13) Siegel, M. M. etal.Anal. Chem. 1991, 63,2470-81. (14) Jimenez, C. R et al./. Neurochem. 1194, Catherine Fenselau is professor or biochemistry at the University ofMaryland-Baltimore 62,404-7. (15) Vestling, M. M.; Kelly, M. A; Fenselau, C. County. Addresssorrespondence to Fenselau Rapid Commun. Mass Spectrom. 1994,8,at the Department of Chemistry and Bio786-90. chemistry, University ofMaryland-Baltimore (16) Anderson, J. S.; Svensson, B.; Roepstorif County, 1000 Hilltop Circle, Baltimore, MM P. Nature Biotech. 1996,14,449-57. 21250 (
[email protected])) (17) Youngquist, R S.; Fuentes, G. R; Lacey, Analytical
Chemistry
HYDRANAL® reagents at a better price. Call today: Aldrich 800-558-9160 414-273-3850 Sigma 800-325-3010
314-771-5750
Riedel-deHaen
SIGMA-ALDRICH RESEARCH Riedel-de Haen is a member of the Sigma-Aldrich family providing Laboratory Chemicals and Reagents for Research and Analysis
CIRCLE 4 ON READER SERVICE CARD News & Features, November 1, 1997 6 6 5 A
