Faculty Bibliography

Acetylation is a well-studied posttranslational modification that has been associated with a broad spectrum of biological processes, notably gene regulation. Many studies have contributed to our knowledge of the enzymology underlying acetylation, including efforts to understand the molecular mechanism of substrate recognition by several acetyltransferases, but traditional experiments to determine intrinsic features of substrate site specificity have proven challenging. Here, we combine experimental methods with clustering analysis of protein sequences to predict protein acetylation based on the sequence characteristics of acetylated lysines within histones with our unique prediction tool PredMod. We define a local amino acid sequence composition that represents potential acetylation sites by implementing a clustering analysis of histone and nonhistone sequences. We show that this sequence composition has predictive power on 2 independent experimental datasets of acetylation marks. Finally, we detect acetylation for selected putative substrates using mass spectrometry, and report several nonhistone acetylated substrates in budding yeast. Our approach, combined with more traditional experimental methods, may be useful for identifying acetylated substrates proteome-wide

Disulfide-rich peptide venoms from animals such as snakes, spiders, scorpions, and certain marine snails represent one of nature's great diversity libraries of bioactive molecules. The various species of marine cone shells have alone been estimated to produce >50,000 distinct peptide venoms. These peptides have stimulated considerable interest because of their ability to potently alter the function of specific ion channels. To date, only a small fraction of this immense resource has been characterized because of the difficulty in elucidating their primary structures, which range in size between 10 and 80 aa, include up to 5 disulfide bonds, and can contain extensive posttranslational modifications. The extraordinary complexity of crude venoms and the lack of DNA databases for many of the organisms of interest present major analytical challenges. Here, we describe a strategy that uses mass spectrometry for the elucidation of the mature peptide toxin components of crude venom samples. Key to this strategy is our use of electron transfer dissociation (ETD), a mass spectrometric fragmentation technique that can produce sequence information across the entire peptide backbone. However, because ETD only yields comprehensive sequence coverage when the charge state of the precursor peptide ion is sufficiently high and the m/z ratio is low, we combined ETD with a targeted chemical derivatization strategy to increase the charge state of cysteine-containing peptide toxins. Using this strategy, we obtained full sequences for 31 peptide toxins, using just 7% of the crude venom from the venom gland of a single cone snail (Conus textile)

Individual posttranslational modifications (PTMs) on histones have well established roles in certain biological processes, notably transcriptional programming. Recent genomewide studies describe patterns of covalent modifications, such as H3 methylation and acetylation at promoters of specific target genes, or 'bivalent domains,' in stem cells, suggestive of a possible combinatorial interplay between PTMs on the same histone. However, detection of long-range PTM associations is often problematic in antibody-based or traditional mass spectrometric-based analyses. Here, histone H3 from a ciliate model was analyzed as an enriched source of transcriptionally active chromatin. Using a recently developed mass spectrometric approach, combinatorial modification states on single, long N-terminal H3 fragments (residues 1-50) were determined. The entire modification status of intact N termini was obtained and indicated correlations between K4 methylation and H3 acetylation. In addition, K4 and K27 methylation were identified concurrently on one H3 species. This methodology is applicable to other histones and larger polypeptides and will likely be a valuable tool in understanding the roles of combinatorial patterns of PTMs

Histone post-translational modifications have been recently intensely studied owing to their role in regulating gene expression. Here, we describe protocols for the characterization of histone modifications in both qualitative and semiquantitative manners using chemical derivatization and tandem mass spectrometry. In these procedures, extracted histones are first derivatized using propionic anhydride to neutralize charge and block lysine residues, and are subsequently digested using trypsin, which, under these conditions, cleaves only the arginine residues. The generated peptides can be easily analyzed using online LC-electrospray ionization-tandem mass spectrometry to identify the modification site. In addition, a stable isotope-labeling step can be included to modify carboxylic acid groups allowing for relative quantification of histone modifications. This methodology has the advantage of producing a small number of predicted peptides from highly modified proteins. The protocol should take approximately 15-19 h to complete, including all chemical reactions, enzymatic digestion and mass spectrometry experiments

Mass spectrometry has played an integral role in the identification of proteins and their post-translational modifications (PTM). However, analysis of some PTMs, such as phosphorylation, sulfonation, and glycosylation, is difficult with collision-activated dissociation (CAD) since the modification is labile and preferentially lost over peptide backbone fragmentation, resulting in little to no peptide sequence information. The presence of multiple basic residues also makes peptides exceptionally difficult to sequence by conventional CAD mass spectrometry. Here we review the utility of electron transfer dissociation (ETD) mass spectrometry for sequence analysis of post-translationally modified and/or highly basic peptides. Phosphorylated, sulfonated, glycosylated, nitrosylated, disulfide bonded, methylated, acetylated, and highly basic peptides have been analyzed by CAD and ETD mass spectrometry. CAD fragmentation typically produced spectra showing limited peptide backbone fragmentation. However, when these peptides were fragmented using ETD, peptide backbone fragmentation produced a complete or almost complete series of ions and thus extensive peptide sequence information. In addition, labile PTMs remained intact. These examples illustrate the utility of ETD as an advantageous tool in proteomic research by readily identifying peptides resistant to analysis by CAD. A further benefit is the ability to analyze larger, non-tryptic peptides, allowing for the detection of multiple PTMs within the context of one another

BACKGROUND: Complete genome annotation is a necessary tool as Anopheles gambiae researchers probe the biology of this potent malaria vector. RESULTS: We reannotate the A. gambiae genome by synthesizing comparative and ab initio sets of predicted coding sequences (CDSs) into a single set using an exon-gene-union algorithm followed by an open-reading-frame-selection algorithm. The reannotation predicts 20,970 CDSs supported by at least two lines of evidence, and it lowers the proportion of CDSs lacking start and/or stop codons to only approximately 4%. The reannotated CDS set includes a set of 4,681 novel CDSs not represented in the Ensembl annotation but with EST support, and another set of 4,031 Ensembl-supported genes that undergo major structural and, therefore, probably functional changes in the reannotated set. The quality and accuracy of the reannotation was assessed by comparison with end sequences from 20,249 full-length cDNA clones, and evaluation of mass spectrometry peptide hit rates from an A. gambiae shotgun proteomic dataset confirms that the reannotated CDSs offer a high quality protein database for proteomics. We provide a functional proteomics annotation, ReAnoXcel, obtained by analysis of the new CDSs through the AnoXcel pipeline, which allows functional comparisons of the CDS sets within the same bioinformatic platform. CDS data are available for download. CONCLUSION: Comprehensive A. gambiae genome reannotation is achieved through a combination of comparative and ab initio gene prediction algorithms

Tri-methylation of histone H3 lysine 9 is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging and heterochromatin formation. Here we show that HP1alpha, -beta, and -gamma are released from chromatin during the M phase of the cell cycle, even though tri-methylation levels of histone H3 lysine 9 remain unchanged. However, the additional, transient modification of histone H3 by phosphorylation of serine 10 next to the more stable methyl-lysine 9 mark is sufficient to eject HP1 proteins from their binding sites. Inhibition or depletion of the mitotic kinase Aurora B, which phosphorylates serine 10 on histone H3, causes retention of HP1 proteins on mitotic chromosomes, suggesting that H3 serine 10 phosphorylation is necessary for the dissociation of HP1 from chromatin in M phase. These findings establish a regulatory mechanism of protein-protein interactions, through a combinatorial readout of two adjacent post-translational modifications: a stable methylation and a dynamic phosphorylation mark

A method for rapid sequencing of intact proteins simultaneously from the N and C termini (1-2 s) with online chromatography is described and applied to the characterization of histone H3.1 posttranslational modifications and the identification of an additional member of the H2A gene family. Proteins are converted to gas-phase multiply charged positive ions by electrospray ionization and then allowed to react with fluoranthene radical anions. Electron transfer to the multiply charged protein promotes random dissociation of the N-Calpha bonds of the protein backbone. Multiply charged fragment ions are then deprotonated in a second ion/ion reaction with the carboxylate anion of benzoic acid. The m/z values for the resulting singly and doubly charged ions are used to read a sequence of 15-40 aa at both the N and C termini of the protein. This information, with the measured mass of the intact protein, is used to search protein or nucleotide databases for possible matches, detect posttranslational modifications, and determine possible splice variants

Histone phosphorylation has long been associated with condensed mitotic chromatin; however, the functional roles of these modifications are not yet understood. Histones H1 and H3 are highly phosphorylated from late G2 through telophase in many organisms, and have been implicated in chromatin condensation and sister chromatid segregation. However, mutational analyses in yeast and biochemical experiments with Xenopus extracts have demonstrated that phosphorylation of H1 and H3 is not essential for such processes. In this study, we investigated additional histone phosphorylation events that may have redundant functions to H1 and H3 phosphorylation during mitosis. We developed an antibody to H4 and H2A that are phosphorylated at their respective serine 1 (S1) residues and found that H4S1/H2AS1 are highly phosphorylated in the mitotic chromatin of worm, fly, and mammals. Mitotic H4/H2A phosphorylation has similar timing and localization as H3 phosphorylation, and closely correlates with the chromatin condensation events during mitosis. We also detected a lower level of H4/H2A phosphorylation in 5-bromo-2-deoxyuridine-positive S-phase cells, which corroborates earlier studies that identified H4S1 phosphorylation on newly synthesized histones during S-phase. The evolutionarily conserved phosphorylation of H4/H2A during the cell cycle suggests that they may have a dual purpose in chromatin condensation during mitosis and histone deposition during S-phase