Identification of 2D-gel proteins: A comparison of MALDI/TOF peptide mass mapping to μ LC-ESI tandem mass spectrometry

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and μLC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by μLC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTipC18 and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using μLC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. μLC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by μLC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for ∼70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.

[1]  David E. Misek,et al.  Isoelectric focusing nonporous RP HPLC: a two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI-TOF mass spectrometry. , 2000, Analytical chemistry.

[2]  Joan C. Han,et al.  A procedure for quantitative determination of tris(2-carboxyethyl)phosphine, an odorless reducing agent more stable and effective than dithiothreitol. , 1994, Analytical biochemistry.

[3]  J. Yates,et al.  Large-scale analysis of the yeast proteome by multidimensional protein identification technology , 2001, Nature Biotechnology.

[4]  A. Żarnecki.  Global analysis of , 1999, hep-ph/9904334.

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

[6]  M R Wilkins,et al.  Strategy for protein isoform identification from expressed sequence tags and its application to peptide mass fingerprinting , 2001, Proteomics.

[7]  Richard D. Smith,et al.  Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses. , 2001, Analytical chemistry.

[8]  H. Wenschuh,et al.  The dominance of arginine-containing peptides in MALDI-derived tryptic mass fingerprints of proteins. , 1999, Analytical chemistry.

[9]  S. Gygi,et al.  Quantitative analysis of complex protein mixtures using isotope-coded affinity tags , 1999, Nature Biotechnology.

[10]  Christian Scheler,et al.  Peptide mass fingerprint sequence coverage from differently stained proteins on two‐dimensional electrophoresis patterns by matrix assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) , 1998, Electrophoresis.

[11]  J. Yates.  Mass spectrometry and the age of the proteome. , 1998, Journal of mass spectrometry : JMS.

[12]  P Berndt,et al.  Reliable automatic protein identification from matrix‐assisted laser desorption/ionization mass spectrometric peptide fingerprints , 1999, Electrophoresis.

[13]  Rovshan G Sadygov,et al.  Code developments to improve the efficiency of automated MS/MS spectra interpretation. , 2002, Journal of proteome research.

[14]  R D Appel,et al.  Improving protein identification from peptide mass fingerprinting through a parameterized multi‐level scoring algorithm and an optimized peak detection , 1999, Electrophoresis.

[15]  J. Yates,et al.  Identifying the major proteome components of Haemophilus influenzae type‐strain NCTC 8143 , 1997, Electrophoresis.

[16]  Ruedi Aebersold,et al.  Quantitative Proteome Analysis by Solid-phase Isotope Tagging and Mass Spectrometry Beads Photocleavable Linker Isotope Tag Reactive Group , 2022 .

[17]  John R. Yates,et al.  A STRATEGY FOR THE IDENTIFICATION OF PROTEINS LOCALIZED TO SUBCELLULAR SPACES : APPLICATION TO E. COLI PERIPLASMIC PROTEINS , 1997 .

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

[19]  N. Anderson,et al.  Analytical techniques for cell fractions. XXI. Two-dimensional analysis of serum and tissue proteins: multiple isoelectric focusing. , 1978, Analytical biochemistry.

[20]  N. Anderson,et al.  Analytical techniques for cell fractions , 1969 .

[21]  B. Monsarrat,et al.  Mapping and Structural Dissection of Human 20 S Proteasome Using Proteomic Approaches* , 2002, Molecular & Cellular Proteomics.

[22]  Thomas E. Creighton,et al.  Protein structure : a practical approach , 1997 .

[23]  P. Roepstorff,et al.  Localization of an O-glycosylated site in the recombinant barley alpha-amylase 1 produced in yeast and correction of the amino acid sequence using matrix-assisted laser desorption/ionization mass spectrometry of peptide mixtures. , 1994, Biological mass spectrometry.

[24]  N. Anderson,et al.  The Human Plasma Proteome , 2002, Molecular & Cellular Proteomics.

[25]  X. Yao,et al.  Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus. , 2001, Analytical chemistry.

[26]  Edmond J. Breen,et al.  Automatic Poisson peak harvesting for high throughput protein identification , 2000, Electrophoresis.

[27]  Kathleen R. Cho,et al.  A 2-D liquid separations/mass mapping method for interlysate comparison of ovarian cancers. , 2002, Analytical chemistry.

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

[29]  J. Yates,et al.  Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low-femtomole level. , 1997, Analytical chemistry.

[30]  A. Podtelejnikov,et al.  Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[31]  John I. Clark,et al.  Shotgun identification of protein modifications from protein complexes and lens tissue , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Peter R. Baker,et al.  Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching. , 1999, Analytical chemistry.

[33]  N. Anderson,et al.  Quantitative reproducibility of measurements from Coomassie Blue‐stained two‐dimensional gels: Analysis of mouse liver protein patterns and a comparison of BALB/c and C57 strains , 1985 .

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

[35]  D. N. Perkins,et al.  Probability‐based protein identification by searching sequence databases using mass spectrometry data , 1999, Electrophoresis.

[36]  J. Yates,et al.  An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database , 1994, Journal of the American Society for Mass Spectrometry.

[37]  J. Yates,et al.  Direct analysis of protein complexes using mass spectrometry , 1999, Nature Biotechnology.

[38]  A. Podtelejnikov,et al.  Identification of the components of simple protein mixtures by high-accuracy peptide mass mapping and database searching. , 1997, Analytical chemistry.

[39]  R. Aebersold,et al.  Mass spectrometry in proteomics. , 2001, Chemical reviews.

[40]  M. Ünlü,et al.  Difference gel electrophoresis. A single gel method for detecting changes in protein extracts , 1997, Electrophoresis.

[41]  K. Markides,et al.  Column switching in capillary liquid chromatography-tandem mass spectrometry for the quantitation of pg/ml concentrations of the free basic drug tolterodine and its active 5-hydroxymethyl metabolite in microliter volumes of plasma. , 1998, Journal of chromatography. A.

[42]  B. Chait,et al.  ProFound: an expert system for protein identification using mass spectrometric peptide mapping information. , 2000, Analytical chemistry.

[43]  S. Carr,et al.  Examination of micro-tip reversed-phase liquid chromatographic extraction of peptide pools for mass spectrometric analysis. , 1998, Journal of chromatography. A.

[44]  J. Yates,et al.  Global analysis of a "simple" proteome: Methanococcus jannaschii. , 2002, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[45]  David L. Tabb,et al.  A proteomic view of the Plasmodium falciparum life cycle , 2002, Nature.

[46]  M. Münchbach,et al.  Quantitation and facilitated de novo sequencing of proteins by isotopic N-terminal labeling of peptides with a fragmentation-directing moiety. , 2000, Analytical chemistry.

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

[48]  A. Burlingame,et al.  Protein identification by in-gel digestion, high-performance liquid chromatography, and mass spectrometry: Peptide analysis by complementary ionization techniques , 2001, Journal of the American Society for Mass Spectrometry.

[49]  J. Yates,et al.  Automated identification of amino acid sequence variations in proteins by HPLC/microspray tandem mass spectrometry. , 2000, Analytical chemistry.

[50]  N G Anderson,et al.  Analytical techniques for cell fractions. XXII. Two-dimensional analysis of serum and tissue proteins: multiple gradient-slab gel electrophoresis. , 1978, Analytical biochemistry.

[51]  F. Cross,et al.  Accurate quantitation of protein expression and site-specific phosphorylation. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[52]  J. Yates,et al.  Protein identification at the low femtomole level from silver-stained gels using a new fritless electrospray interface for liquid chromatography-microspray and nanospray mass spectrometry. , 1998, Analytical biochemistry.