Presentation on theme: "The ABRF Edman Sequencing Research Group 2007 Study: Results of Deacetylation Procedures used to Determine the N-Terminal Sequence of a Blocked Protein."— Presentation transcript:
The ABRF Edman Sequencing Research Group 2007 Study: Results of Deacetylation Procedures used to Determine the N-Terminal Sequence of a Blocked Protein B.S. Hampton 1, R.S. Thoma 2, D. Brune 3, N.D. Denslow 4, P. Hunziker 5, K.D. Linse 6, J. Pohl 7, J.W. Leone 8 1 University of Maryland School of Medicine, Baltimore, MD USA, 2 Monsanto Co., St. Louis, MO, USA, 3 Arizona State University, Tempe, AZ, USA 4 University of Florida, Gainsville, FL, USA, 5 University of Zurich, Zurich, Switzerland, 6 University of Texas, Austin, TX, USA, 7 Emory University, Atlanta, GA, USA, 8 Pfizer Inc., St. Louis, MO, USA Amino Acid Sequence Assignment Accuracy Conclusions Results of Deblocking Methods The Edman Sequencing Research Group (ESRG) designs studies on the use of Edman degradation for protein and peptide analysis. These studies provide a means for participating laboratories to compare their analyses against a benchmark of those from other laboratories that provide this valuable service. The main purpose of the ESRG 2007 study was to analyze techniques for chemically deblocking the N-terminus of an N- terminally acetylated protein thus making it susceptible to Edman degradation. The test sample that was distributed to participating laboratories was bovine Histone H4, a protein that is known to have an N-acetylserine at it’s N-terminus. The sample tube contained 450 picomoles; considered to be enough protein for three sequence determinations. The participating laboratories were asked to deblock the protein, determine the sequence of the first 20 amino acids including any modified amino acids encountered, and conduct a database search to identify the protein. To aid participants in this study, references from the literature, as well as two procedures used by the ESRG with some success were provided. Participants were not limited to using these procedures and were encouraged to use any procedures they have developed for the purpose of deblocking an N-acetylated protein. An important aspect of this study was to compile the treatments and techniques that were used for deblocking the ESRG 2007 sample to ascertain their effectiveness. Therefore, participants were asked to provide detailed reporting of their experimental protocols and techniques to ensure that successful strategies for deblocking can be reproduced by the Edman sequencing community. Details about instruments and parameters used in the analysis were also collected. Introduction Bovine histone H4 was purified by a combination of cation exchange and reversed phase chromatography using modifications of Couppez(1) and Sarg(2) in a manner that allowed the use of those columns and reagents that were on hand in the laboratory. Essentially, a crude preparation of histones from bovine thymus was purchased from Worthington and was dissolved in 25mM triethylamine phosphate pH 3, 6M urea, 0.3M guanidine-HCl and subjected to cation exchange chromatography on a polysulphoethyl-aspartamede column. Proteins were eluted in this buffer with a gradient of guanidine-HCl from 0.3M to 2.0M. SDS-PAGE was used to assay these fractions for the presence histone H4 based on the characteristic migration pattern of histones on a coomassie blue stained gel (3,4) where histones H3, H2B, H2A, and H4 are the predominant stained bands with histone H4 migrating the fastest. Those fractions containing histone H4 were pooled and purified further by reversed phase chromatography on a Phenomenex Jupiter C18 column. Final purity was achieved after rechromatography on a Higgins Analytical TARGA C8 column. Purity was estimated to be at least 95 percent based on the coomassie blue staining of approximately 5 ug of the preparation loaded onto an SDS-PAGE gel. The protein concentration was determined by absorbance at 280 nm after applying the extinction coefficient that was calculated from the unmodified amino acid sequence (http://www.expasy.org/tools/protparam.html). Additionally, the protein concentration was also determined by amino acid compositional analysis following vapor phase acid hydrolysis for 1.5 hr at 150°C and analysis on a Hitachi L-8800 with post column ninhydrin derivitization. Identity of this protein as histone H4 was confirmed following digestion with trypsin and analysis of the resulting peptides by LC MS/MS and searching the Bos taurus (ENSEMBL) database using the search algorithm !XTandem (5). Aliquots of 8 µl were dried by rotary evaporation and stored at -80°C until distributed to the requesting laboratories. Methods for deblocking histone H4 were based upon published studies of Wellner (6) and Bergman(7). Two methods were used by the ESRG to validate that this protein could indeed be deblocked. These methods involved exposure of the protein to vapor, or liquid phase trifluoroacetic acid either in a test tube, or on the sequencing instrument itself. A cycle printout of the on instrument gas phase deblocking method was posted on the ABRF web site (8) Prior to acid treatment the protein was immobilized on either a PVDF membrane or polybrene treated glass fiber filter according to established procedures. The histone H4 preparation was characterized further to provide evidence that the N- terminus is blocked. One hundred fifty picomoles of histone H4 was immobilized on a PVDF membrane and subjected directly to Edman sequencing to show the percent of the protein preparation that was refractory to Edman chemistry. Additionally, histone H4 was digested with endoproteinase Asp-N and the peptide corresponding to the N-terminus of the protein was analyzed by MALDI MS, and finally the intact protein was analyzed by an ESI QTOF-MS and the mass of the intact protein determined after deconvoluting the multiply charged envelope of peaks with MaxEnt software. Materials and Methods Nature of the Test Sample Tricine-SDS-PAGE Position* Modification 1 N-acetylserine 3 Symmetric dimethylarginine (alternate by similarity) 5 N6-acetyllysine (By similarity) 8 N6-acetyllysine (By similarity) 12 N6-acetyllysine (By similarity 16 N6-acetyllysine 20 N6,N6-dimethyllysine(alternate) 20 N6-methyllysine (alternate) 47 Phosphoserine (By similarity *Numbering excludes the initiator methionine. Amino acid sequence and feature table of bovine histone H4 Taken from http://www.expasy.org/uniprot/h4_bovin/ ESI QTOF MS Analysis SGRGKGGKGLGKGGAKRHR K VLRDNIQGITKPAIRRLARR GGVKRISGLIYEETRGVLKV FLENVIRDAVTYTEHAKRKT VTAMDVVYALKRQGRTLYGF GG References 1.Couppez, M., Martin-Ponthieu, A., Sautière, P. (1987) Histone H4 from Cuttlefish Testis is Sequentially Acetylated JBC 262, 2854-2860. 2.Sarg, B., Koutzamani, E., Helliger, W., Rundquist, I. (2002) Postsynthetic Trimethylation of Histone H4 at Lysine 20 in Mammalian Tissues is Associated with Aging JBC 277, 39195-39201 3.Ball, D., Slaughter, C., Hensley, P., Garrard, W., (1983) Amino Acid Sequence of the N-terminal Domain of Calf Thymus histone H2A.Z FEBS Lett. 154, 166-170 4.Sarg, B., Helliger, W., Talasz, H., Koutzamani, E., Lindner, H. (2004) Histine H4 Hyperacetylation Precludes Histone H4 Lysine 20 Trimethylation JBC 279, 53458-53464 5.Web site for !Xtandem database search: http://www.thegpm.org/ 6.Wellner, D., Panneerselvam, C. and Horecker, B.L. (1990) Sequencing of peptides and proteins with blocked N-terminal amino acids: N-Acetylserine or N-acetylthreonine Proc. Natl Acad. Sci.USA 87, 1947-1949. 7.Bergman, T., Gheorghe, M.T., Hjelmqvist, L. and Jörnvall, H. (1996) Alcoholytic deblocking of N-terminally acetylated peptides and proteins for sequence analysis FEBS Lett. 390, 199-202. 8.Web site for on-sequencer deblocking cycle: http://www.abrf.org/ResearchGroups/EdmanSequencing/Studies/DeblockCycle.pdf/ Results The primary goal of this study was to assess the ability of the Edman sequencing community to deblock the N-terminus of histone H4 (a protein known to have N-acetylserine at its N-terminus), sequence the first 20 residues and identify any modified amino acids that may be present. A source of histone H4 was not readily available so the ESRG purified bovine histone H4 from a crude preparation to high purity as judged by SDS-PAGE. It was demonstrated that the N-terminus was blocked following Edman sequencing of the untreated sample where no amino acid sequence was observed (data not shown). Analysis of the untreated protein by ESI QTOFMS and deconvolution of the multiply charged envelope of peaks showed masses of (11306.50, 11348.00, 11364.50, 11404.50 and 11446.50) indicating that the histone H4 present in the sample contained a number of posttranslational modifications consistent with a combination of acetylation, methylation, and oxidation of amino acid side chains. The mass 11236.15 corresponding to the calculated mass of the unmodified protein was absent from these spectra. Since the untreated protein was refractory to Edman degradation it was assumed that the N-terminus was also modified. All but one participant used TFA to deblock the N-terminus. Nine used a mixture of TFA and methanol, nine used gas phase delivery on the sequencing instrument, and three used TFA. Fifteen of the twenty three participants who returned data were able to deblock histone H4 and determine enough of the N-terminal amino acid sequence to identify the protein by a database search. Serine was positively identified by eleven, and tentatively identified by five participants in cycle one. Of the ten participants who provided amino acid sequence beyond sixteen cycles, one participant observed acetylated lysine in position 16 and methylated lysine in position 20, nine were able to identify an acetylated lysine in position 16, and one participant observed acetylated lysine in positions 5, 8, 12, and 16. The addition of methanol to the TFA used to deblock the protein improved the ability to positively identify serine in cycle one and to identify acetylated lysine in cycle sixteen. Of those participants who used either gas or liquid phase TFA without methanol, three positively identified serine and two identified acetylated lysine in cycles one and sixteen respectively, whereas seven participants positively identified serine and five identified acetylated lysine in cycles one and sixteen respectively when methanol was included in the TFA. The ESRG tested four cycles of deblocking with gas phase delivery of TFA containing twenty percent hexafluoroisopropanol and demonstrated some improvement in overall yield and ease of identifying N-terminal serine and the modified lysines in the sequence. Analysis of internal cleavages of histone H4 that were generated during the acid deblocking procedure was done by collating the amino acids reported to be observed in each cycle and matching them with the sequences that would be generated by Ser-N, Thr-N or Asp-C cleavage of the protein. This analysis revealed four “hot spots” of internal cleavage at threonine in positions 30, 54, 80, and 96. Threonine in these positions is flanked on either side by a charged amino acid. Data from only one participant indicated cleavage at the single internal serine, and evidence for cleavage at threonine in position 71 was not apparent in any of the participants data. Both of these sites are flanked on both sides by non-polar amino acids. Initial Yield Calculation for Histone H4 Sequence N.R. indicates no data was reported for any amino acid - (dash) indicates data was reported for an incorrectly assigned PTH-amino acid Comparison of Gas Phase Deblocking 1 2 3 75 kD - 50 kD - 37 kD - 25 kD - 20 kD - 15 kD - 10 kD - *Indicates one, four or nine cycles of gas phase deblocking **Four cycles of gas phase deblockiing with TFA:HFIP (80:20) Summary of Deprotection Methods and Initial Yields A majority of participating laboratories were able to deblock the protein and obtain enough amino acid sequence information to identify it as histone H4. Identification of the modified amino acids in the sequence was less successful as less than half of the participants were able to identify acetyl- lysine-16, the major site of lysine acetylation observed in this preparation. Both liquid and gas phases of TFA were used successfully to deblock histone H4. In accordance with a previous study by Bergman et. al. (7), adding methanol or in one case hexaflurorisopropanol, to the TFA improved the deblocking results based on the increased number of participants that identified serine in cycle one and acetyllysine in cycle sixteen. ESI MS analysis of the intact protein by the ESRG revealed five distinct mass-resolved forms. Based on these data and the following observations that a mass corresponding to unmodified histone H4 was not observed, the untreated protein was refractory to Edman degradation and susceptibility to Edman degradation occurred only following an acid treatment procedure, it was deduced that the blocking group could be an acetyl moiety. Therefore, removal of the blocking group during the acid treatment procedures could have occurred by the acid-catalyzed N O shift reaction mechanism. However, the precise nature of the blocking group itself was not determined in this study. The deblocking efficiency of histone H4 was less than 10 percent for most participants and the ESRG combined, but the initial yields varied significantly between participant's laboratories. Multiple experiments performed by the ESRG showed similar initial yields compared to those obtained by many of the participants, however, the initial yields in the ESRG experiments were fairly consistent with each other even though slight variations in the procedure were employed. In addition to cleavage of the N-terminal blocking group at serine-1, internal peptide bond cleavage was observed primarily at the N-terminal side of threonine, and this cleavage appeared to occur more frequently when threonine was flanked by a charged amino acid. These specific internal peptide bond cleavages may be occurring by the same N O shift reaction mechanism that also deblocks the protein but seems to occur at a reduced rate allowing one to discern the N-terminal sequence. Thanks to all the participating laboratories for taking the time to analyze the test sample and sending in their results. Without their participation, this effort would not have been successful. Thanks to the American Peptide Society (www.americanpeptidesociety.org) and the Protein Society (www.proteinsociety.org) for announcing the ESRG2007 Study to their members. Thanks also to Renee Crawford and Olga Stuchlik for removing identifiers from the responding laboratories. Acknowledgments Lane 1 - BioRad Precision Plus Standards Lane 2 - Worthington histones Lane 3 - purified histone H4. * The mass 11236.15 corresponding to the mass of the unmodified amino acid sequence was not observed for this sample. ** Delta mass is calculated by subtracting the mass calculated from the unmodified amino acid sequence of bovine histone H4 from the observed mass. Ac = Acetyl; diMe = diMethyl; OxMet = oxidized Methionine * Mean values were calculated after omitting zero values and those values above the theoretical maximum.