title: Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Analysis of Peptides.
abstract: Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a simple, effective, and widely used technique for determining the mass of biomolecules, with exceptional capabilities for mass analysis of peptides. Relative to other ionization techniques, MALDI-MS offers a high tolerance of salts and other common buffer components. MALDI-MS ionization of peptides results in predominantly singly charged, protonated ion species (M+H(+)), rendering analysis relatively straightforward. MALDI-TOF/TOF-MS instruments enable true tandem MS/MS fragmentation of peptide ions, facilitating peptide sequencing and identity. Routine detection limits for peptides are in the low parts per million range. This unit will focus on the application of MALDI-MS to the analysis of peptides on time-of-flight (TOF) mass spectrometers. Instrument configuration and calibration, matrix options, and sample preparation are discussed. Finally, this unit describes methods for obtaining sequence information on peptides by tandem mass spectrometry.
authors.affiliation: Department of Protein Chemistry, Genentech, South San Francisco, California.
authors.forename: Wendy
authors.initials: W
authors.lastname: Sandoval
mesh_terms: D010455:Peptides / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods* / Q000592:standards
chemical_list: D010455:Peptides
keywords: MALDI; mass spectrometry; matrix; matrix assisted laser desorption/ionization; peptide analysis; time-of-flight
references.citation: Bahr, U. , Karas, M. , and Hillenkamp, F. 1994. Analysis of biopolymers by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Frensenius J. Anal. Chem. 348:783-791.
references.citation: Beavis, R.C. 1992. Matrix-assisted ultraviolet laser desorption: Evolution and principles. Org. Mass Spectrom. 27:653-659.
references.citation: Beavis, R.C. , Chaudhary, T. , and Chait, B.T. 1992. α-Cyana-4-hydroxy cinnamic acid as a matrix for matrix-assisted laser desorption mass spectrometry. Org. Mass Spectrom. 27:156-158.
references.citation: Billeci, T.M. and Stults, J.T. 1993. Tryptic mapping of recombinant proteins by matrix-assisted laser desorption/ionization mass spectrometry. Anal. Chem. 65:1709-1716.
references.citation: Billing, A. , Kessler, J. , Revets, D. , Sausy, A. , Schmitz, S. , Barra, C. , and Muller, C. 2014. Proteome profiling of virus-host interactions of wild type and attenuated measles virus strains. J. Proteomics 108:325-226.
references.citation: Bodnar, W. , Blackburn, K. , Krise, J. , and Moseley, A. 2003. Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage. 14:971-979.
references.citation: Brown, R.S. and Lennon, J.J. 1995. Mass resolution improvement by incorporation of pulsed ion extraction in a matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometer. Anal. Chem. 67:1998-2003.
references.citation: Caprioli, R. , Farmer, T. , and Gile, J. 1997. Molecular imaging of biological samples: Localization of peptides and proteins using MALDI-TOF MS. Anal. Chem. 69:4751-4760.
references.citation: Cohen, S.L. and Chait, B.T. 1996. Influence of matrix solution conditions on the MALDI-MS analysis of peptides and proteins. Anal. Chem. 68:31-37.
references.citation: Colby, S.M. , King, T.B. , and Reilly, J.P. 1994. Improving the resolution of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry by exploiting the correlation between ion position and velocity. Rapid Commun. Mass Spectrom. 8:865-868.
references.citation: Crimmins, D.L. , Saylor, M. , Rush, J. , and Thoma, R.S. 1995. Facile, in situ matrix-assisted laser desorption ionization-mass spectrometry analysis and assignment of disulfide pairings in heteropeptide molecules. Anal. Biochem. 226:355-361.
references.citation: Demeure, K. , Quinton, L. , Gabelica, V. , and De Pauw, E. 2007. Rational selection of the optimum MALDI matrix for top down proteomics by in-source decay. Anal. Chem. 79:8678-8685.
references.citation: Eigenbrot, C. , Ultsch, M. , Lipari, T. , Moran, P. , Lin, J. , Ganesan, R. , Quan, C. , Tom, J. , Sandoval, W. , van Lookeren Campagne, M. , and Kirchhofer, D. 2012. Structural and functional analysis of HtrA1 and its subdomains. Structure 20:1040-1050.
references.citation: Fabris, D. , Vestling, M.M. , Cordero, M.M. , Doroshenko, V.M. , Cotter, R.J. , and Fenselau, C. 1995. Sequencing electroblotted proteins by tandem mass spectrometry. Rapid Commun. Mass Spectrom. 9:1051-1055.
references.citation: Gharahdaghi, F. , Kirchner, M. , Fernandez, J. , and Mische, S.M. 1996. Peptide-mass profiles of polyvinylidene difluoride-bound proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in the presence of nonionic detergents. Anal. Biochem. 233:94-99.
references.citation: Gorman, J.J. , Wallis, T.P. , and Pitt, J.J. 2002. Protein disulfide bond determination by mass spectrometry. Mass Spectrom. Rev. 21: 183-216.
references.citation: Henzel, W.J. , Billeci, T.M. , Stults, J.T. , Wong, S.C. , Grimley, C. , and Watanabe, C. 1993. Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc. Natl. Acad. Sci. U.S.A. 90:5011-5015.
references.citation: Hillenkamp, F. , Karas, M. , Beavis, R.C. , and Chait, B.T. 1991. Matrix-assisted laser desorption/ionization mass spectrometry of biopolymers. Anal. Chem. 63:1193A-1203A.
references.citation: Huberty, M.C. , Vath, J.E. , Yu, W. , and Martin, S.A. 1993. Site-specific carbohydrate identification in recombinant proteins using MALDI-TOF MS. Anal. Chem. 65:2791-2800.
references.citation: Huwiler, K. , Mosher, D. , and Vestling, M. 2003. Optimizing the MALDI-TOF-MS observation of peptides containing disulfide bonds. J. Biomol. Techn. 14:289-297.
references.citation: Karas, M. and Hillenkamp, F. 1988. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal. Chem. 60:2299-2301.
references.citation: Karas, M. , Ehring, H. , Nordoff, E. , Stahl, B. , Strupat, K. , Hillenkamp, F. , Grehl, M. , and Krebs, B. 1993. Matrix-assisted laser desorption/ionization mass spectrometry with additives to 2,5-dihydrobenzoic acid. Org. Mass Spectrom. 28:1476-1481.
references.citation: Kaufmann, R. 1995. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry: A novel analytical tool in molecular biology and biotechnology. J. Biotechnol. 41:155-175.
references.citation: Keough, T. , Takigiku, R. , Lacey, M.P. , and Purdon, M. 1992. Matrix-assisted laser desorption mass spectrometry of protein isolated by capillary zone electrophoresis. Anal. Chem. 64:1594-1600.
references.citation: Kjellstrom, S. and Jensen, O.N. 2004. Phosphoric acid as a matrix additive for MALDI MS analysis of phosphopeptides and phosphoproteins. Anal. Chem. 76:5109-5117.
references.citation: Lecchi, P. and Caprioli, R.M. 1996. Matrix-assisted laser desorption mass spectrometry for peptide mapping. In New Methods in Peptide Mapping for the Characterization of Proteins ( W.S. Hancock , ed.) pp. 219-240. CRC Press, Boca Raton, Fla.
references.citation: Li, K.W. , Hoek, R.M. , Smith, F. , Jimenez, C.R. , van der Schors, R.C. , Veelen, P.A.V. , Chen, S. , van der Greef, J. , Parish, D.C. , Benjamin, P.R. , and Geraerts, W.P.M. 1994. Direct peptide profiling by mass spectrometry of single identified neurons reveals complex neuropeptide-processing pattern. J. Biol. Chem. 269:30288-30292.
references.citation: Liao, P.-C. , Leykam, J. , Andrews, P.C. , Gage, D.A. , and Allison, J. 1994. An approach to locate phosphorylation sites in a phosphoprotein: Mass mapping by combining specific enzymatic degradation with matrix-assisted laser desorption/ionization mass spectrometry. Anal. Biochem. 219:9-20.
references.citation: Mann, M. 1994. Sequence database searching by mass spectrometric data. In Microcharacterization of Proteins ( R. Kellner , F. Lottspeich , and H.E. Meyer , eds.) pp. 223-245. VCH Publishers, New York.
references.citation: Mann, M. , Hojrup, P. , and Roepstorff, P. 1993. Use of mass spectrometric molecular weight information to identify proteins in sequence databases. Biol. Mass Spectrom. 22:338-345.
references.citation: Medzihradszky, K. , Campbell, J. , Baldwin, M. , Falick, A. , Juhasz, P. , Vestal, M. , Burlingame, A. 2000. The characteristics of peptide collision induced dissociation using a high performance MALDI-TOF/TOF tandem mass spectrometer. Anal. Chem. 72:552-558.
references.citation: Mock, K.K. , Sutton, C.W. , and Cottrell, J.S. 1992. Sample immobilization protocols for matrix-assisted laser desorption mass spectrometry. Rapid Commun. Mass Spectrom. 6:233-238.
references.citation: Morelle, W. , Faid, V. , Chirat, F. , and Michalski, J.C. 2009. Analysis of N- and O-linked glycans from glycoproteins using MALDI-TOF mass spectrometry. Methods Mol. Biol. 534:5-21.
references.citation: Nelson, R.W. , Dogruel, D. , Krone, F.R. , and Williams, P. 1995. Peptide characterization using bioreactive mass spectrometer probe tips. Rapid Commun. Mass Spectrom. 9:1380-1385.
references.citation: Papac, D.I. , Hoyes, J. , and Tomer, K.B. 1994. Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry. Protein Sci. 3:1485-1492.
references.citation: Pappin, D.J.C. , Hojrup, P. , and Bleasby, A.J. 1993. Rapid identification of proteins by peptide-mass fingerprinting. Curr. Biol. 3:327-332.
references.citation: Patterson, D.H. , Tarr, G.E. , Regnier, F.E. , and Martin, S.A. 1995. C-terminal ladder sequencing via matrix-assisted laser desorption mass spectrometry coupled with carboxypeptidase Y time-dependent and concentration-dependent digestions. Anal. Chem. 67:3971-3978.
references.citation: Patterson, S.D. 1995. Matrix-assisted laser-desorption/ionization mass spectrometric approaches for the identification of gel-separated proteins in the 5-50 pmol range. Electrophoresis 16:1104-1114.
references.citation: Patterson, S.D. and Katta, V. 1994. Prompt fragmentation of disulfide-linked peptides during matrix-assisted laser desorption ionization mass spectrometry. Anal. Chem. 66:3727-3732.
references.citation: Robertson, J.G. , Adams, G.W. , Medzihradszky, K.F. , Burlingame, A.L. , and Villafranca, J.J. 1994. Complete assignment of disulfide bonds in bovine dopamine β-hyroxylase. Biochemistry 33:11563-11575.
references.citation: Rosinke, B. , Strupat, K. , Hillenkamp, F. , Rosenbusch, J. , Dencher, N. , Krüger, U. , and Galla, H.J. 1995. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of membrane proteins and non-covalent complexes. J. Mass Spectrom. 30:1462-1468.
references.citation: Rüdiger, A. , Rüdiger, M. , Weber, K. , and Schomburg, D. 1995. Characterization of post-translational modifications of brain tubulin by matrix-assisted laser desorption/ionization mass spectrometry: Direct one-step analysis of a limited subtilisin digest. Anal. Biochem. 224:532-537.
references.citation: Sechi, S. and Chait, B. 1998. Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification. Anal. Chem. 70:5150-5158.
references.citation: Shevchenko, A. , Wilm, M. , and Mann, M. 1996. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem. 68:850-858.
references.citation: Stahl, B. , Klabunde, T. , Witzel, H. , Krebs, B. , Steup, M. , Karas, M. , and Hillenkamp, F. 1994. The oligosaccharides of the Fe(III)-Zn(II) purple acid phosphatase of the red kidney bean. Eur. J. Biochem. 220:321-330.
references.citation: Strupat, K. , Karas, M. , and Hillenkamp, F. 1992. 2,5-Dihydroxybenzoic acid: A new matrix for laser desorption-ionization mass spectrometry. Int. J. Mass Spectrom. Ion Processes. 111:89-102.
references.citation: Stults, J.T. 1995. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Curr. Opin. Struct. Biol. 5:691-698.
references.citation: Sutton, C.W. , O'Neill, J.A. , and Cottrell, J.S. 1994. Site-specific characterization of glycoprotein carbohydrates by exoglycosidase digestion and laser desorption mass spectrometry. Anal. Biochem. 218:34-46.
references.citation: Talbo, G. and Mann, M. 1994. Distinction between phosphorylated and sulfated peptides by matrix-assisted laser desorption/ionization reflector mass spectrometry at the subpicomole level. In Techniques in Protein Chemistry V ( J.W. Crabb , ed.) pp. 105-113. Academic Press, San Diego.
references.citation: Tanaka, K. , Waki, H. , Ido, Y. , Akita, S. , Yoshida, Y. , and Yoshida, T. 1988. Protein and polymer analyses up to m/z 100,000 by laser ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 2:151-153.
references.citation: Thiede, B. , Wittmann-Liebold, B. , Bienert, M. , and Krause, E. 1995. MALDI-MS for C-terminal sequence determination of peptides and proteins degraded by carboxypeptidase Y and P. FEBS Lett. 357:65-69.
references.citation: vanVeelen, P.A. , Tjaden, U.R. , van der Greef, J. , Ingendoh, A. , and Hillenkamp, F. 1993. Off-line coupling of capillary electrophoresis with matrix-assisted laser desorption mass spectrometry. J. Chromatogr. 647:367-374.
references.citation: Vestal, M.L. , Juhasz, P. , and Martin, S.A. 1995. Delayed extraction matrix-assisted laser desorption time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 9:1044-1050.
references.citation: Vorm, O. and Mann, M. 1994. Improved mass accuracy in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of peptides. J. Am. Soc. Mass Spectrom. 5:955-958.
references.citation: Vorm, O. , Roepstorff, P. , and Mann, M. 1994. Improved resolution and very high sensitivity in MALDI TOF of matrix surfaces made by fast evaporation. Anal. Chem. 66:3281-3287.
references.citation: Walker, K.L. , Chiu, R.W. , Monnig, C.A. , and Wilkins, C.L. 1995. Off-line coupling of capillary electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Anal. Chem. 67:4197-4204.
references.citation: Wang, J. , Chen, R. , and Li, L. 2008. MALDI-MS sample preparation using paraffin wax film. Anal. Chem. 80:491-500.
references.citation: Whittal, R.M. and Li, L. 1995. High-resolution matrix-assisted laser desorption/ionization in a linear time-of-flight mass spectrometer. Anal. Chem. 67:1950-1954.
references.citation: Woods, A.S. , Huang, A.Y.C. , Cotter, R.J. , Pasternack, G.R. , Pardoll, D.M. , and Jaffee, E.M. 1995. Simplified high-sensitivity sequencing of a major histocompatibility complex class I-associated immunoreactive peptide using matrix-assisted laser desorption/ionization mass spectrometry. Anal. Biochem. 226:15-25.
references.citation: Xiang, F. and Beavis, R.C. 1994. A method to increase contaminant tolerance in protein matrix-assisted laser desorption/ionization by the fabrication of thin protein-doped polycrystalline films. Rapid Commun. Mass Spectrom. 8:199-204.
references.citation: Yates, J. , Ruse, C. , and Nakorchevsky, A. 2009. Proteomics by mass spectrometry: Approaches, advances, and applications. Biomed. Engin. 11:49-79.
references.citation: Yip, T. and Hutchens, T.W. 1993. Protein phosphorylation: Sequence-specific identification of in vivo phosphorylation sites by MALDI-TOF mass spectrometry. In Techniques in Protein Chemistry IV ( R.H. Angeletti , ed.) pp. 201-210. Academic Press, San Diego.
references.citation: Zaluzec, E.J. , Gage, D.A. , and Watson, J.T. 1994a. Quantitative assessment of cysteine and cystine in peptides and proteins following organomercurial derivatization and analysis by matrix-assisted laser desorption ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 5:359-366.
references.citation: Zaluzec, E.J. , Gage, D.A. , Allison, J. , and Watson, J.T. 1994b. Direct matrix-assisted laser desorption ionization mass spectrometric analysis of proteins immobilized on nylon-based membranes. J. Am. Soc. Mass Spectrom. 5:230-237.
references.citation: Zavalin, A. , Todd, E.M. , Rawhouser, P.D. , Yang, J. , Norris, J.L. , and Caprioli, R.M. 2012. Direct imaging of single cells and tissue at sub-cellular spatial resolution using transmission geometry MALDI MS. J. Mass Spectrom. 47:1473-1481.
references.citation: Zhao, Y. and Chait, B.T. 1994. Protein epitope mapping by mass spectrometry. Anal. Chem. 66:3723-3726.
references.citation: Zhou, J. , Ens, W. , Poppe-Schriemer, N. , Standing, K.G. , and Westmore, J.B. 1993. Cleavage of interchain disulfide bonds following matrix-assisted laser desorption. Int. J. Mass Spectrom. Ion Processes 126:115-122.
references.citation: Zhu, X. and Papayannopoulos, I. 2003. Improvement in the detection of low concentration protein digests on a MALDI-TOF/TOF workstation by reducing alpha-cyano-4-hydroxycinnamic acid adduct ions. J. Biomol. Technol. 4:298-307.
references.citation: Zuberovic, A. , Wetterhall, M. , Hanrieder, J. , and Bergquist, J. 2009. CE MALDI-TOF/TOF MS for multiplexed quantification of proteins in human ventricular cerebrospinal fluid. Electrophoresis 30:1836-1843.