Rapid mass spectrometric identification of proteins from two‐dimensional polyacrylamide gels after in gel proteolytic digestion

We report a rapid method for identifying proteins resolved by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) using matrix‐assisted laser desorption ionization‐mass spectrometry (MALDI‐MS). In‐gel digestion was performed in a way such that the volume ratio of trypsin solution to gel plug was quantitatively controlled to promote reproducible digestion and to maximize the digestion yield. To make the digestion samples more compatible with MALDI‐MS, the volatile salt ammonium bicarbonate in the digestion buffer was largely removed prior to peptide extraction. Samples of mixed tryptic peptides from in‐gel digestion were used without purification to obtain molecular weights by MALDI‐MS with α‐cyano, 4‐hydroxy‐cinnamic acid as the matrix. Modifications of MALDI sample loading procedures improved the detetion sensitivity by one half to one order of magnitude. The peptide mass peaks in MALDI‐MS spectra were distinguished from those of impurities by using several types of controls, and masses were corrected by using trypsin autodigestion fragments as internal calibration standards. Two different peptidematching computer programs were used to interrogate sequence databases and identify proteins. Identification was enhanced by generation of orthogonal data sets (by using different proteases) and by including experimental values of isoelectric point (pI) and molecular weight to exclude false entries in the candidate lists. Approximately 1% of the material from a spot was used in each sample loading, and nine protein spots from rat liver 2‐D PAGE gels were identified correctly, as judged by comparison with identification results previously obtained from Edman sequencing. A previously identified low‐abundance spot was not identified by MALDI‐MS, presumably because there was insufficient material in a single gel. The sample handling procedure reported here should permit us to identify many 2‐D PAGE protein spots of medium abundance.

[1]  Peter Roepstorff,et al.  Improved resolution and very high sensitivity in MALDI TOF of matrix surfaces made by fast evaporation , 1994 .

[2]  R. Aebersold,et al.  Analytical and micropreparative peptide mapping by high performance liquid chromatography/electrospray mass spectrometry of proteins purified by gel electrophoresis , 1993, Protein science : a publication of the Protein Society.

[3]  B. Chait,et al.  Influence of matrix solution conditions on the MALDI-MS analysis of peptides and proteins. , 1996, Analytical chemistry.

[4]  M. Mann,et al.  Automation of micro‐preparation and enzymatic cleavage of gel electrophoretically separated proteins , 1995, FEBS letters.

[5]  R. Nelson,et al.  Rapid tryptic mapping using enzymatically active mass spectrometer probe tips. , 1995, Analytical chemistry.

[6]  C. Watanabe,et al.  Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[7]  A. Burlingame,et al.  Rapid mass spectrometric peptide sequencing and mass matching for characterization of human melanoma proteins isolated by two-dimensional PAGE. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[8]  D. Brune,et al.  Alkylation of cysteine with acrylamide for protein sequence analysis. , 1992, Analytical biochemistry.

[9]  E. Müller,et al.  Identification of human myocardial proteins separated by two‐dimensional electrophoresis using an effective sample preparation for mass spectrometry , 1996, Electrophoresis.

[10]  M. Mann,et al.  Electrospray ionization for mass spectrometry of large biomolecules. , 1989, Science.

[11]  P Ferrara,et al.  In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. , 1992, Analytical biochemistry.

[12]  S. Patterson,et al.  Matrix‐assisted laser‐desorption/ionization mass spectrometric approaches for the identification of gel‐separated proteins in the 5–50 pmol range , 1995, Electrophoresis.

[13]  J. Bonaventura,et al.  Acrylamide in polyacrylamide gels can modify proteins during electrophoresis. , 1994, Analytical biochemistry.

[14]  N. Anderson,et al.  An updated two‐dimensional gel database of rat liver proteins useful in gene regulation and drug effect studies , 1991 .

[15]  R. Aebersold,et al.  Mass spectrometric approaches for the identification of gel‐separated proteins , 1995, Electrophoresis.

[16]  A. Shevchenko,et al.  Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry , 1996, Nature.

[17]  L. D. Ward,et al.  Two‐dimensional electrophoretic analysis of proteins expressed by normal and cancerous human crypts: Application of mass spectrometry to peptide‐mass fingerprinting , 1994, Electrophoresis.

[18]  G. Siuzdak The emergence of mass spectrometry in biochemical research. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[19]  M. Wilm,et al.  Error-tolerant identification of peptides in sequence databases by peptide sequence tags. , 1994, Analytical chemistry.

[20]  G. Gonnet,et al.  Protein identification by mass profile fingerprinting. , 1993, Biochemical and biophysical research communications.

[21]  J N Weinstein,et al.  A protein expression database for the molecular pharmacology of cancer , 1997, Electrophoresis.

[22]  U. Hellman,et al.  Improvement of an "In-Gel" digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. , 1995, Analytical biochemistry.

[23]  E Carafoli,et al.  Protein identification in DNA databases by peptide mass fingerprinting , 1994, Protein science : a publication of the Protein Society.

[24]  C. Fenselau,et al.  Poly(vinylidene difluoride) membranes as the interface between laser desorption mass spectrometry, gel electrophoresis, and in situ proteolysis , 1994 .

[25]  Cottrell Js,et al.  Protein identification by peptide mass fingerprinting. , 1994 .

[26]  P. Højrup,et al.  Rapid identification of proteins by peptide-mass fingerprinting , 1993, Current Biology.

[27]  M. Karas,et al.  Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. , 1988, Analytical chemistry.

[28]  A. Shevchenko,et al.  Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. , 1996, Analytical chemistry.

[29]  Joël Vandekerckhove,et al.  The human keratinocyte two‐dimensional gel protein database (update 1992): Towards an integrated approach to the study of cell proliferation, differentiation and skin diseases , 1992, Electrophoresis.

[30]  P. Højrup,et al.  Use of mass spectrometric molecular weight information to identify proteins in sequence databases. , 1993, Biological mass spectrometry.

[31]  F. Neidhardt,et al.  Genomically linked cellular protein databases derived from two‐dimensional polyacrylamide gel electrophoresis , 1989, Electrophoresis.

[32]  N G Anderson,et al.  Twenty years of two‐dimensional electrophoresis: Past, present and future , 1996, Electrophoresis.

[33]  P. Roepstorff,et al.  Identification of proteins in polyacrylamide gels by mass spectrometric peptide mapping combined with database search. , 1994, Biological mass spectrometry.

[34]  S. Patterson From electrophoretically separated protein to identification: strategies for sequence and mass analysis. , 1994, Analytical biochemistry.

[35]  Amos Bairoch,et al.  Human liver protein map: A reference database established by microsequencing and gel comparison , 1992, Electrophoresis.

[36]  J. Celis,et al.  A qualitative and quantitative protein database approach identifies individual and groups of functionally related proteins that are differentially regulated in simian virus 40 (SV40) transformed human keratinocytes: An overview of the functional changes associated with the transformed phenotype , 1994, Electrophoresis.

[37]  M. Qian,et al.  Peptide Mapping by CNBr Degradation on a Nitrocellulose Membrane with Analysis by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry , 1995 .

[38]  G. S. Johnson,et al.  An Information-Intensive Approach to the Molecular Pharmacology of Cancer , 1997, Science.