Enhanced top-down characterization of histone post-translational modifications

Post-translational modifications (PTMs) of core histones work synergistically to fine tune chromatin structure and function, generating a so-called histone code that can be interpreted by a variety of chromatin interacting proteins. We report a novel online two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) platform for high-throughput and sensitive characterization of histone PTMs at the intact protein level. The platform enables unambiguous identification of 708 histone isoforms from a single 2D LC-MS/MS analysis of 7.5 µg purified core histones. The throughput and sensitivity of comprehensive histone modification characterization is dramatically improved compared with more traditional platforms.     

Strategy for comprehensive identification of post-translational modifications in cellular proteins, including low abundant modifications: application to glyceraldehyde-3-phosphate dehydrogenase

Post-translational modifications (PTMs) play key roles in the regulation of biological functions of proteins. Although some progress has been made in identifying several PTMs using existing approaches involving a combination of affinity-based enrichment and mass spectrometric analysis, comprehensive identification of PTMs remains a challenging problem in proteomics because of the dynamic complexities of PTMs in vivo and their low abundance. We describe here a strategy for rapid, efficient, and comprehensive identification of PTMs occurring in biological processes in vivo. It involves a selectively excluded mass screening analysis (SEMSA) of unmodified peptides during liquid chromatography-electrospray ionization-quadrupole-time-of-flight tandem mass spectrometry (LC-ESI-q-TOF MS/MS) through replicated runs of a purified protein on two-dimensional gel. A precursor ion list of unmodified peptides with high mass intensities was obtained during the initial run followed by exclusion of these unmodified peptides in subsequent runs. The exclusion list can grow as long as replicate runs are iteratively performed. This enables the identifications of modified peptides with precursor ions of low intensities by MS/MS sequencing. Application of this approach in combination with the PTM search algorithm MODi to GAPDH protein in vivo modified by oxidative stress provides information on multiple protein modifications (19 types of modification on 42 sites) with >92% peptide coverage and the additional potential for finding novel modifications, such as transformation of Cys to Ser. On the basis of the information of precursor ion m/z, quantitative analysis of PTM was performed for identifying molecular changes in heterogeneous protein populations. Our results show that PTMs in mammalian systems in vivo are more complicated and heterogeneous than previously reported. We believe that this strategy has significant potential because it permits systematic characterization of multiple PTMs in functional proteomics.     

The intracellular distribution and function of the high mobility group chromosomal proteins

This brief review provides a framework for discussing current approaches being used to determine the cellular localization and function of the high mobility group chromosomal (HMG) proteins. The four main constituents of this group (HMG 1, 2, 14, 17) are present in all four eukaryotic kingdoms, have a relatively well conserved primary sequence and contain several functional domains which enable them to interact with DNA, histones and other components of the genome. The evolutionary conservation in the primary and tertiary structure as well as the observed correlations between cell phenotype and quantitative changes in protein levels and in post-synthesis modifications suggests that these proteins are components obligatory for proper cellular function. Proteins HMG 1, 2 are DNA-binding proteins which can distinguish between various types of single-stranded regions of the genome. Proteins HMG 14, 17 may be involved in maintaining specific chromatin regions in particular conformations. The data available presently suggests that these proteins are important structural elements of chromatin and chromosomes.     

Lysine methylation: beyond histones

Posttranslational modifications (PTMs) of histone proteins, such as acetylation, methylation, phosphorylation, and ubiquitylation, play essential roles in regulating chromatin dynamics. Combinations of different modifications on the histone proteins, termed 'histone code' in many cases, extend the information potential of the genetic code by regulating DNA at the epigenetic level. Many PTMs occur on non-histone proteins as well as histones, regulating protein-protein interactions, stability, localization, and/or enzymatic activities of proteins involved in diverse cellular processes. Although protein phosphorylation, ubiquitylation, and acetylation have been extensively studied, only a few proteins other than histones have been reported that can be modified by lysine methylation. This review summarizes the current progress on lysine methylation of non-histone proteins, and we propose that lysine methylation, like phosphorylation and acetylation, is a common PTM that regulates proteins in diverse cellular processes.     

Sequence tag scanning: a new explorative strategy for recognition of unexpected protein alterations by nanoelectrospray ionization-tandem mass spectrometry

Protein analysis by database search engines using tandem mass spectra is limited by the presence of unexpected protein modifications, sequence isoforms which may not be in the protein databases, and poor quality tandem mass spectrometry (MS/MS) of low abundance proteins. The analysis of expected protein modifications can be efficiently addressed by precursor ion scanning. However, it is limited to modifications that show such a characteristic loss in a peptide independent manner. We observed that proline and aspartic acid induced backbone fragmentation is accompanied by a low intensity signal for loss of H3PO4 for several pSer- or pThr-phosphopeptides. We describe here the use of peptide-specific fragments that can be used after a protein was identified to allow in-depth characterization of modifications and isoforms. We consider high abundance fragments formed by cleavage at the C-terminal side of aspartic acid, at the N-terminal side of proline and low mass ions such as a2, b2, b3, y1, y2, and y3. The MS/MS dataset is filtered for each sequence tag of interest by an in silico precursor ion scan. The resulting extracted ion traces are then combined by multiplication to increase specificity. Since the strategy is based on common peptide segments which are shared by different isoforms of peptides it can be applied to the analysis of any post-translational modification or sequence variants of a protein. This is demonstrated for the cases of serine and threonine phosphorylation, histone H1 acetylation and the spotting of multiple H1 isoforms.     

Mass spectrometric mapping of linker histone H1 variants reveals multiple acetylations, methylations, and phosphorylation as well as differences between cell culture and tissue

Posttranslational modifications of histones are involved in regulation of chromatin structure and gene activity. Whereas the modifications of the core histones H2A, H2B, H3, and H4 have been extensively studied, our knowledge of H1 modifications remained mainly limited to its phosphorylation. Here we analyzed the composition of histone H1 variants and their modifications in two human cell lines and nine mouse tissues. Use of a hybrid linear ion trap-orbitrap mass spectrometer facilitated assignment of modifications by high resolution and low ppm mass accuracy for both the precursor and product mass spectra. Across different tissues we identified a range of phosphorylation, acetylation, and methylation sites. We also mapped sites of ubiquitination and report identification of formylated lysine residues. Interestingly many of the mapped modifications are located within the globular domain of the histones at sites that are thought to be involved in binding to nucleosomal DNA. Investigation of mouse tissue in addition to cell lines uncovered a number of interesting differences. For example, whereas methylation sites are frequent in tissues, this type of modification was much less abundant in cultured cells and escaped detection. Our study significantly extends the known spectrum of linker histone variability.     

Analysis of posttranslational modifications of proteins by tandem mass spectrometry

Protein activity and turnover is tightly and dynamically regulated in living cells. Whereas the three-dimensional protein structure is predominantly determined by the amino acid sequence, posttranslational modification (PTM) of proteins modulates their molecular function and the spatial-temporal distribution in cells and tissues. Most PTMs can be detected by protein and peptide analysis by mass spectrometry (MS), either as a mass increment or a mass deficit relative to the nascent unmodified protein. Tandem mass spectrometry (MS/MS) provides a series of analytical features that are highly useful for the characterization of modified proteins via amino acid sequencing and specific detection of posttranslationally modified amino acid residues. Large-scale, quantitative analysis of proteins by MS/MS is beginning to reveal novel patterns and functions of PTMs in cellular signaling networks and biomolecular structures.     

Proteomic profiling of intact proteins using WAX-RPLC 2-D separations and FTICR mass spectrometry

We investigated the combination of weak anion exchange (WAX) fractionation and on-line reversed-phase liquid chromatography (RPLC) separation using a 12 T FTICR mass spectrometer for the detection of intact proteins from a Shewanella oneidensis MR-1 cell lysate. This work aimed at optimizing intact protein detection for profiling proteins at a level that incorporates their modification state. A total of 715 intact proteins were detected, and the combined results from the WAX fractions and the unfractionated cell lysate were aligned using LC-MS features to facilitate protein abundance measurements. Protein identifications and post-translational modifications were assigned for approximately 10% of the detected proteins by comparing intact protein mass measurements to proteins identified in peptide MS/MS analysis of an aliquot of the same fraction. Intact proteins were also detected for S. oneidensis lysates obtained from cells grown on 13C-, 15N-depleted media under aerobic and sub-oxic conditions. The strategy can be readily applied for measuring differential protein abundances and provides a platform for high-throughput selection of biologically relevant targets for further characterization.     

Beyond linker histones and high mobility group proteins: global profiling of perchloric acid soluble proteins

Extraction with HClO(4) provides an easy method for efficient enrichment of both histone H1 and HMG proteins from a variety of tissues. Usually, the histone and the HMG proteins are the most abundant components of the extracts, however, other proteins have frequently been observed but only seldom studied in more detail. Here we describe a study aimed at global characterization of HClO(4) extractable proteins from breast cancer cell lines. We report identification of 150 unique proteins by liquid chromatography tandem mass spectrometry including almost all major histone H1 variants and canonical members of the HMG protein families. In the extracts, diverse proteins with HMG-like amino acid composition were identified and their post-translational modifications were mapped. Importantly, those include multiple proteins known or supposed to be related to cell proliferation and cancer. Since purification of these proteins as well as low abundant variants of histone and HMG proteins is difficult due to their metabolic instability, characterization of these proteins from crude extracts can facilitate studies aimed at better understanding of their function.     

VEMS 3.0: algorithms and computational tools for tandem mass spectrometry based identification of post-translational modifications in proteins

Protein and peptide mass analysis and amino acid sequencing by mass spectrometry is widely used for identification and annotation of post-translational modifications (PTMs) in proteins. Modification-specific mass increments, neutral losses or diagnostic fragment ions in peptide mass spectra provide direct evidence for the presence of post-translational modifications, such as phosphorylation, acetylation, methylation or glycosylation. However, the commonly used database search engines are not always practical for exhaustive searches for multiple modifications and concomitant missed proteolytic cleavage sites in large-scale proteomic datasets, since the search space is dramatically expanded. We present a formal definition of the problem of searching databases with tandem mass spectra of peptides that are partially (sub-stoichiometrically) modified. In addition, an improved search algorithm and peptide scoring scheme that includes modification specific ion information from MS/MS spectra was implemented and tested using the Virtual Expert Mass Spectrometrist (VEMS) software. A set of 2825 peptide MS/MS spectra were searched with 16 variable modifications and 6 missed cleavages. The scoring scheme returned a large set of post-translationally modified peptides including precise information on modification type and position. The scoring scheme was able to extract and distinguish the near-isobaric modifications of trimethylation and acetylation of lysine residues based on the presence and absence of diagnostic neutral losses and immonium ions. In addition, the VEMS software contains a range of new features for analysis of mass spectrometry data obtained in large-scale proteomic experiments. Windows binaries are available at http://www.yass.sdu.dk/.     

Identification of protein complexes that bind to histone H3 combinatorial modifications using super-SILAC and weighted correlation network analysis

The large number of chemical modifications that are found on the histone proteins of eukaryotic cells form multiple complex combinations, which can act as recognition signals for reader proteins. We have used peptide capture in conjunction with super-SILAC quantification to carry out an unbiased high-throughput analysis of the composition of protein complexes that bind to histone H3K9/S10 and H3K27/S28 methyl-phospho modifications. The accurate quantification allowed us to perform Weighted correlation network analysis (WGCNA) to obtain a systems-level view of the histone H3 histone tail interactome. The analysis reveals the underlying modularity of the histone reader network with members of nuclear complexes exhibiting very similar binding signatures, which suggests that many proteins bind to histones as part of pre-organized complexes. Our results identify a novel complex that binds to the double H3K9me3/S10ph modification, which includes Atrx, Daxx and members of the FACT complex. The super-SILAC approach allows comparison of binding to multiple peptides with different combinations of modifications and the resolution of the WGCNA analysis is enhanced by maximizing the number of combinations that are compared. This makes it a useful approach for assessing the effects of changes in histone modification combinations on the composition and function of bound complexes.     

P-Mod: an algorithm and software to map modifications to peptide sequences using tandem MS data

The discovery of unanticipated protein modifications is one of the most challenging problems in proteomics. Whereas widely used algorithms such as Sequest and Mascot enable mapping of modifications when the mass and amino acid specificity are known, unexpected modifications cannot be identified with these tools. We have developed an algorithm and software called P-Mod, which enables discovery and sequence mapping of modifications to target proteins known to be represented in the analysis or identified by Sequest. P-Mod matches MS/MS spectra to peptide sequences in a search list. For spectra of modified peptides, P-Mod calculates mass differences between search peptide sequences and MS/MS precursors and localizes the mass shift to a sequence position in the peptide. Because modifications are detected as mass shifts, P-Mod does not require the user to guess at masses or sequence locations of modifications. P-Mod uses extreme value statistics to assign p value estimates to sequence-to-spectrum matches. The reported p values are scaled to account for the number of comparisons, so that error rates do not increase with the expanded search lists that result from incorporating potential peptide modifications. Combination of P-Mod searches from multiple LC-MS/MS analyses and multiple samples revealed previously unreported BSA modifications, including a novel decarboxymethylation or D-->G substitution at position 579 of the protein. P-Mod can serve a unique role in the identification of protein modifications both from exogenous and endogenous sources and may be useful for identifying modified protein forms as biomarkers for toxicity and disease processes.     

Liquid chromatography mass spectrometry profiling of histones

Here we describe the use of reverse-phase liquid chromatography mass spectrometry (RPLC-MS) to simultaneously characterize variants and post-translationally modified isoforms for each histone. The analysis of intact proteins significantly reduces the time of sample preparation and simplifies data interpretation. LC-MS analysis and peptide mass mapping have previously been applied to identify histone proteins and to characterize their post-translational modifications. However, these studies provided limited characterization of both linker histones and core histones. The current LC-MS analysis allows for the simultaneous observation of all histone PTMs and variants (both replacement and bulk histones) without further enrichment, which will be valuable in comparative studies. Protein identities were verified by the analysis of histone H2A species using RPLC fractionation, AU-PAGE separation and nano-LC-MS/MS.     

Attomole detection of in vivo protein targets of benzene in mice: evidence for a highly reactive metabolite

Modified proteins were detected in liver and bone marrow of mice following treatment with [(14)C]benzene. Stained sections were excised from one-dimensional and two-dimensional gels and converted to graphite to enable (14)C/(13)C ratios to be measured by accelerator mass spectrometry. Protein adducts of benzene or its metabolites were indicated by elevated levels of (14)C. A number of proteins were identified by in-gel proteolysis and conventional mass spectrometric methods with the low molecular weight proteins identified including hemoglobin and several histones. The incorporation of (14)C was largely proportional to the density of gel staining, giving little evidence that these proteins were specific targets for selective labeling. This was also true for individual histones subfractionated with Triton-acid-urea gels. A representative histone, H4, was isolated and digested with endopeptidase Asp-N, and the resulting peptides were separated by high performance liquid chromatography. (14)C levels in collected fractions were determined, and the peptides were identified by conventional mass spectrometry. The modifications were distributed throughout the protein, and no particular amino acids or groups of amino acids were identified as selective targets. Thus chemical attack by one or more benzene metabolites upon histones was identified and confirmed, but the resulting modifications appeared to be largely nonspecific. This implies high reactivity toward proteins, enabling such attack to occur at multiple sites within multiple targets. It is not known to what extent, if any, the modification of the core histones may contribute to the carcinogenicity of benzene.