Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification

  • Hongying Zhong
  • Sandra L. Marcus
  • Liang LiEmail author


Simple and efficient digestion of proteins, particularly hydrophobic membrane proteins, is of significance for comprehensive proteome analysis using the bottom-up approach. We report a microwave-assisted acid hydrolysis (MAAH) method for rapid protein degradation for peptide mass mapping and tandem mass spectrometric analysis of peptides for protein identification. It uses 25% trifluoroacetic acid (TFA) aqueous solution to dissolve or suspend proteins, followed by microwave irradiation for 10 min. This detergent-free method generates peptide mixtures that can be directly analyzed by liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) without the need of extensive sample cleanup. LC-MALDI MS/MS analysis of the hydrolysate from 5 µg of a model transmembrane protein, bacteriorhodopsin, resulted in almost complete sequence coverage by the peptides detected, including the identification of two posttranslational modification sites. Cleavage of peptide bonds inside all seven transmembrane domains took place, generating peptides of sizes amenable to MS/MS to determine possible sequence errors or modifications within these domains. Cleavage specificity, such as glycine residue cleavage, was observed. Terminal peptides were found to be present in relatively high abundance in the hydrolysate, particularly when low concentrations of proteins were used for MAAH. It was shown that these peptides could still be detected from MAAH of bacteriorhodopsin at a protein concentration of 1 ng/µl or 37 fmol/µl. To evaluate the general applicability of this method, it was applied to identify proteins from a membrane protein enriched fraction of cell lysates of human breast cancer cell line MCF7. With one-dimensional LC-MALDI MS/MS, a total of 119 proteins, including 41 membrane-associated or membrane proteins containing one to 12 transmembrane domains, were identified by MS/MS database searching based on matches of at least two peptides to a protein.


MALDI Acid Hydrolysis Microwave Irradiation Protein Identification Human Breast Cancer Cell Line 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Supplementary material

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  1. 1.
    Wu, C. C.; Yates, J. R. III. The Application of Mass Spectrometry to Membrane Proteomics. Nat. Biotechnol 2003, 21, 262–267.CrossRefGoogle Scholar
  2. 2.
    Rabilloud, T. Membrane Proteins Ride Shotgun. Nat. Biotechnol. 2003, 21, 508–510.CrossRefGoogle Scholar
  3. 3.
    Han, D. K.; Eng, J.; Zhou, H.; Aebersold, R. Quantitative Profiling of Differentiation-induced Microsomal Proteins Using Isotope-Coded Affinity Tags and Mass Spectrometry. Nat. Biotechnol. 2001, 19, 946–951.CrossRefGoogle Scholar
  4. 4.
    Norris, J. L.; Porter, N. A.; Caprioli, R. M. Mass Spectrometry of Intracellular and Membrane Proteins Using Cleavable Detergents. Anal. Chem. 2003, 75, 6642–6647.CrossRefGoogle Scholar
  5. 5.
    Blonder, J.; Goshe, M. B.; Moore, R. J.; Pasa-Tolic, L.; Masselon, C. D.; Lipton, M. S.; Smith, R. D. Enrichment of Integral Membrane Proteins for Proteomic Analysis Using Liquid Chromatography-Tandem Mass Spectrometry. J. Prot. Res. 2002, 1, 351–360.CrossRefGoogle Scholar
  6. 6.
    Goshe, M. B.; Blonder, J.; Smith, R. D. Affinity Labeling of Highly Hydrophobic Integral Membrane Proteins for Proteome-Wide Analysis. J. Prot. Res. 2003, 2, 153–161.CrossRefGoogle Scholar
  7. 7.
    Blonder, J.; Conrads, T. P.; Yu, L.; Terunuma, A.; Janini, G. M.; Issaq, H. J.; Vogel, J. C.; Veenstra, T. D. A Detergent-and Cyanogen Bromide-Free Method for Integral Membrane Proteomics: Application to Halobacterium Purple Membranes and the Human Epidermal Membrane Proteome. Proteomics 2004, 4, 31–45.CrossRefGoogle Scholar
  8. 8.
    Ball, L. E.; Oatis, J. E.; Dharmasiri, K.; Busman, M.; Wang, J.; Cowden, L. B.; Galijatovic, A.; Chen, L.; Crouch, R. K.; Knapp, D. R. Mass Spectrometric Analysis of Integral Membrane Proteins: Application to Complete Mapping of Bacteriorhodopsins and Rhodopsin. Prot. Sci. 1998, 7, 758–764.CrossRefGoogle Scholar
  9. 9.
    Ablonczy, Z.; Kono, M.; Crouch, R. K.; Knapp, D. R. Mass Spectrometric Analysis of Integral Membrane Proteins at the Subnanomolar Level: Application to Recombinant Photopigments. Anal. Chem. 2001, 73, 4774–4779.CrossRefGoogle Scholar
  10. 10.
    Washburn, M. P.; Wolters, D.; Yates, J. R. III. Large-Scale Analysis of the Yeast Proteome by Multidimensional Protein Identification Technology. Nat. Biotechnol. 2001, 19, 242–247.CrossRefGoogle Scholar
  11. 11.
    Quach, T. T. T.; Li, N.; Richard, D. P.; Zheng, J.; Keller, B. O.; Li, L. Development and Applications of In-Gel CNBr/Tryptic Digestion Combined with Mass Spectrometry for the Analysis of Membrane Proteins. J. Prot. Res. 2003, 2, 543–552.CrossRefGoogle Scholar
  12. 12.
    Wu, C. C.; MacCoss, M. J.; Howell, K. E.; Yates, J. R. III. A Method for the Comprehensive Proteomic Analysis of Membrane Proteins. Nat. Biotechnol. 2003, 21, 532–538.CrossRefGoogle Scholar
  13. 13.
    Sanger, F.; Thompson, E. O. P. The Amino-acid Sequence in the Glycyl Chain of Insulin: 1. The Identification of Lower Peptides from Partial Hydrolysates. Biochem. J. 1953, 53, 353–366.Google Scholar
  14. 14.
    Sonderegger, P. R.; Jaussi, H.; Gehring, K.; Brunschweiler, K.; Christen, P. Peptide Mapping of Protein Bands from Polyacrylamide Gel Electrophoresis by Chemical Cleavage in Gel Pieces and Re-electrophoresis. Anal. Biochem. 1982, 122, 298–301.CrossRefGoogle Scholar
  15. 15.
    Vanfleteren, J. R.; Raymackers, J. G.; van Bun, S. M.; Meheus, L. A. Peptide Mapping and Microsequencing of Proteins Separated by SDS-PAGE after Limited In Site Acid Hydrolysis. BioTechnique 1992, 12, 550–557.Google Scholar
  16. 16.
    Tsugita, A.; Takamoto, K.; Kamo, M.; Iwadate, H. C-terminal Sequencing of Protein: A Novel Partial Acid Hydrolysis and Analysis by Mass Spectrometry. Eur. J. Biochem 1992, 206, 691–696.CrossRefGoogle Scholar
  17. 17.
    Tsugita, A.; Kamo, M.; Miyazaki, K.; Takayama, M.; Kawakami, T.; Shen, R.; Nozawa, T. Additional Possible Tools for Identification of Proteins on One- or Two-Dimensional Electrophoresis. Electrophoresis 1998, 19, 928–938.CrossRefGoogle Scholar
  18. 18.
    Vorm, O.; Roepstorff, P. Peptide Sequence Information Derived by Partial Acid Hydrolysis and Matrix-Assisted Laser Desportion/Ionization Mass Spectrometry. Biol. Mass Spectrom. 1994, 23, 734–740.CrossRefGoogle Scholar
  19. 19.
    Zubarev, R. A.; Chivanov, V. D.; Hakansson, P.; Sundkvist, B. U. R. Peptide Sequencing by Partial Acid Hydrolysis and High Resolution Plasma Desorption Mass Spectrometry. Rapid Commun. Mass Spectrom. 1994, 8, 906–912.CrossRefGoogle Scholar
  20. 20.
    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.CrossRefGoogle Scholar
  21. 21.
    Li, A.; Sowder, R. C. II; Henderson, L. E.; Moore, S. P.; Garfinfel, D. J.; Fisher, R. J. Chemical Cleavage at Aspartyl Residues for Protein Identification. Anal. Chem 2001, 73, 5395–5402.CrossRefGoogle Scholar
  22. 22.
    Shevchenko, A.; Loboda, A.; Shevchenko, A.; Ens, W.; Standing, K. G. MALDI Quadrupole Time-of-Flight Mass Spectrometry: A Powerful Tool for Proteomic Research. Anal. Chem. 2000, 72, 2132–2141.CrossRefGoogle Scholar
  23. 23.
    Lin, S. H.; Tornatore, P.; Weinberger, S. R.; King, D.; Orlando, R. Limited Acid Hydrolysis as a Means of Fragmenting Proteins Isolated upon Proteinchip Array Surfaces. Eur. J. Mass Spectrom 2001, 7, 131–141.CrossRefGoogle Scholar
  24. 24.
    Ocana, M. F.; Neubert, H.; Przyborowska, A.; Parker, R.; Bramley, P.; Halket, J.; Patel, R. BSE Control: Detection of Gelatine-Derived Peptides in Animal Feed by Mass Spectrometry. Analyst 2004, 129, 111–115.CrossRefGoogle Scholar
  25. 25.
    Giguerre, R. J.; Bray, T. L.; Duncan, S. M. Application of Commercial Microwave Ovens to Organic Synthesis. Tetrahedron Lett. 1986, 27, 4945–4948.CrossRefGoogle Scholar
  26. 26.
    Gedye, R.; Smith, F.; Westway, K.; Ali, H.; Baldisera, L.; Laberge, L.; Rousell, J. The Use of Microwave Ovens for Rapid Organic Synthesis. Tetrahedron Lett. 1986, 27, 279–282.CrossRefGoogle Scholar
  27. 27.
    Kingston, H. M.; Haswell, S. J. Microwave-Enhanced Chemistry: Fundamentals, Sample Preparation, and Applications; ACS: Washington, D. C., 1997.Google Scholar
  28. 28.
    Lew, A.; Krutzik, P. O.; Hart, M. E.; Chamberlin, A. R. Increasing Rates of Reaction: Microwave-Assisted Organic Synthesis for Combinatorial Chemistry. J. Comb. Chem. 2002, 4, 95–105.CrossRefGoogle Scholar
  29. 29.
    Chen, S. T.; Chiou, S. H.; Chu, Y. H.; Wang, K. T. Rapid Hydrolysis of Proteins and Peptides by Means of Microwave Technology and Its Application to Amino Acid Analysis. Int. J. Peptide Protein Res. 1987, 30, 572–576.CrossRefGoogle Scholar
  30. 30.
    Chiou, S. H.; Wang, K. T. Peptide and Protein Hydrolysis by Microwave Irradiation. J. Chromatogr. 1989, 491, 424–431.CrossRefGoogle Scholar
  31. 31.
    Chen, S. T.; Chiou, S. H.; Wang, K. T. Enhancement of Chemical Reactions by Microwave Irradiation. J. Chin. Chem. Soc 1991, 38, 85–91.Google Scholar
  32. 32.
    Chen, S. T.; Tseng, P. H.; Yu, H. M.; Wu, C. Y.; Hsiao, K. F.; Wu, S. H.; Wang, K. T. The Studies of Microwave Effects on the Chemical Reactions. J. Chin. Chem. Soc. 1997, 44, 169–182.Google Scholar
  33. 33.
    Bose, A. K.; Ing, Y. H.; Lavlinskaia, N.; Sareen, C.; Pramanik, B. N.; Bartner, P. L.; Liu, Y. H.; Heimark, L. Microwave Enhanced Akabori Reaction for Peptide Analysis. J. Am. Soc. Mass Spectrom. 2002, 13, 839–850.CrossRefGoogle Scholar
  34. 34.
    Pramanik, B. N.; Ing, Y. H.; Bose, A. K.; Zhang, L. K.; Liu, Y. H.; Ganguly, S. N.; Bartner, P. Rapid Cyclopeptide Analysis by Microwave Enhanced Akabori Reaction. Tetrahedron Lett. 2003, 44, 2565–2568.CrossRefGoogle Scholar
  35. 35.
    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. Prot. Sci. 2002, 11, 2676–2687.CrossRefGoogle Scholar
  36. 36.
    Zhang, B.; McDonald, C.; Li, L. Combining Liquid Chromatography with MALDI Mass Spectrometry Using a Heated Droplet Interface. Anal. Chem. 2004, 76, 992–1001.CrossRefGoogle Scholar
  37. 37.
    Dai, Y. Q.; Whittal, R. M.; Li, L. Two-Layer Sample Preparation: A Method for MALDI-MS Analysis of Complex Peptide and Protein Mixtures. Anal. Chem. 1999, 71, 1087–1091.CrossRefGoogle Scholar
  38. 38.
    Hixson, K. K.; Rodriguez, N.; Camp, D. G. II, Lipton, M. S.; Smith, R. D. Evaluation of Enzymatic Digestion and Liquid Chromatography-Mass Spectrometry Peptide Mapping of the Integral Membrane Protein Bacteriorhodopsin. Electrophoresis 2002, 23, 3224–3232.CrossRefGoogle Scholar
  39. 39.
    Smith, B. J. In The Protein Protocols Handbook, 2nd ed.; Walker, J. M.; Ed Humana Press: Totowa, NJ, 2002; 485–491.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2005

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of AlbertaEdmontonCanada

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