Abstract
Sample preparation is crucial to the success of experiments in biological mass spectrometry. In proteomics, digestion of the proteins into peptides is a key step for “bottom-up” approaches. Often, the use of enzymes requires physiological conditions, producing peptides that must be extracted or further purified before mass analysis. Chemical cleavage reagents offer more flexibility and can be more compatible with downstream mass analysis. Expanding on prior work using acid hydrolysis, proteolysis with matrix-assisted laser desorption ionization (MALDI) matrices is presented. This sample preparation can be performed rapidly with a minimum of reagents and sample handling, but it must first be evaluated in terms of digestion efficiency, missed cleavages, and side reactions before implementation for in-gel digestion and in-solution digestion using minimal volumes of protein. Time courses of acid hydrolysis are shown for protein standards, illustrating the sensitivity of this type of sample preparation, minimization of side reactions, and performance for proteins in mixtures. To illustrate the potential for sensitive detection of a specific protein, MALDI matrix hydrolysis is used to digest a protein immunoprecipitated from cell lysate.
Article PDF
Similar content being viewed by others
References
Russell, W. K.; Park, Z.-Y.; Russell, D. H. Proteolysis in mixed organic-aqueous solvent systems: Applications for peptide mass mapping using mass spectrometry. Anal. Chem. 2001, 73, 2682–2685.
Pramanik, B. N.; Mirza, U. A.; Ing, Y. H.; Liu, Y. H.; Bartner, P. L.; Weber, P. C.; Bose, A. K. Microwave-enhanced enzyme reaction for protein mapping by mass spectrometry: A new approach to protein digestion in minutes. Protein Sci. 2002, 11, 2676–2687.
Juan, H. F.; Chang, S. C.; Huang, H. C.; Chen, S. T. A new application of microwave technology to proteomics. Proteomics 2005, 5, 840–842.
Lin, S. S.; Wu, C. H.; Sun, M. C.; Sun, C. M.; Ho, Y. P. Microwave-assisted enzyme-catalyzed reactions in various solvent systems. J. Am. Soc. Mass Spectrom. 2005, 16, 581–588.
Lin, S.; Lin, Z.; Yao, G.; Deng, C.; Yang, P.; Zhang, X. Development of microwave-assisted protein digestion based on trypsin-immobilized magnetic microspheres for highly efficient proteolysis followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. Rapid Commun. Mass Spectrom. 2007, 21, 3910–3918.
Fountoulakis, M.; Lahm, H.-W. Hydrolysis and amino acid composition of proteins. J. Chromatogr. A 1998, 826, 109–134.
Gobom, J.; Mirgorodskaya, E.; Nordhoff, E.; Hojrup, P.; Roepstorff, P. Use of vapor-phase acid hydrolysis for mass spectrometric peptide mapping and protein identification. Anal. Chem. 1999, 71, 919–927.
Li, A.; Sowder, R. C.; Henderson, L. E.; Moore, S. P.; Garfinkel, D. J.; Fisher, R. J. Chemical cleavage at aspartyl residues for protein identification. Anal. Chem. 2001, 73, 5395–5402.
Hua, L.; Low, T. Y.; Sze, S. K. Microwave-assisted specific chemical digestion for rapid protein identification. Proteomics 2006, 5, 586–591.
Zhong, H.; Marcus, S. L.; Li, L. Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification. J. Am. Soc. Mass Spectrom. 2005, 16, 471–481.
Swatkoski, S.; Gutierrez, P.; Ginter, J.; Petrov, A.; Dinman, J. D.; Edwards, N.; Fenselau, C. Integration of residue-specific acid cleavage into proteomic workflows. J. Proteome Res. 2007, 6, 4525–4527.
Swatkoski, S.; Gutierrez, P.; Wynne, C.; Petrov, A.; Dinman, J. D.; Edwards, N.; Fenselau, C. Evaluation of microwave-accelerated residue-specific acid cleavage for proteomic applications. J. Proteome Res. 2008, 7, 579–586.
Swatkoski, S.; Russell, S. C.; Edwards, N.; Fenselau, C. Rapid chemical digestion of small acid-soluble spore proteins for analysis of Bacillus spores. Anal. Chem. 2006, 78, 181–188.
Swatkoski, S.; Russell, S.; Edwards, N.; Fenselau, C. Analysis of a model virus using residue-specific chemical cleavage and MALDI-TOF mass spectrometry. Anal. Chem. 2007, 79, 654–658.
Wang, N.; Mackenzie, L.; De Souza, A. G.; Zhong, H.; Goss, G.; Li, L. Proteome profile of cytosolic component of zebrafish liver generated by LC-ESI MS/MS combined with trypsin digestion and microwave-assisted acid hydrolysis. J. Proteome Res. 2007, 6, 263–272.
Reported by SciFinder 2007, calculated by Advanced Chemistry Development (ACD/Labs) Software V8.14 for Solaris 1994–2008.
Harris, D. C. Quantitative Chemical Analysis, 3rd ed.; W. H. Freeman and Co: New York, 1991; pp. AP24-AP32.
Palmblad, M.; Cramer, R. Liquid matrix deposition on conductive hydrophobic surfaces for tuning and quantitation in UV-MALDI mass spectrometry. J. Am. Soc. Mass Spectrom. 2007, 18, 693–697.
Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20, 3551–3567.
Lu, D.; Liu, R. Z.; Izumi, V.; Fenstermacher, D.; Haura, E. B.; Koomen, J.; Eschrich, S. A. IPEP: An in silico tool to examine proteolytic peptides for mass spectrometry. Bioinformatics 2008, 24, 2801–2802.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Remily-Wood, E., Dirscherl, H. & Koomen, J.M. Acid hydrolysis of proteins in matrix assisted laser desorption ionization matrices. J Am Soc Mass Spectrom 20, 2106–2115 (2009). https://doi.org/10.1016/j.jasms.2009.07.007
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1016/j.jasms.2009.07.007