Transcriptional states are formed and maintained by the interaction and post-translational modification (PTM) of several chromatin proteins, such as histones and high mobility group (HMG) proteins. Among these, HMGA1a, a small heterochromatin-associated nuclear protein has been shown to be post-translationally modified, and some of these PTMs have been linked to apoptosis and cancer. In cancerous cells, HMGA1a PTMs differ between metastatic and nonmetastatic cells, suggesting the existence of an HMGA1a PTM code analogous to the "histone code." In this study, we expand on current knowledge by comprehensively characterizing PTMs on HMGA1a purified from human cells using both nanoflow liquid chromatography collision activated dissociation mediated Bottom Up and electron-transfer dissociation facilitated middle and Top Down mass spectrometry (MS). We find HMGA1a to be pervasively modified with many types of modifications such as methylation, acetylation, and phosphorylation, including finding novel sites. While Bottom Up MS identified lower level modification sites, Top and Middle Down MS were utilized to identify the most commonly occurring combinatorially modified forms. Remarkably, although we identify several individual modification sites through our Bottom Up and Middle Down MS analyses, we find relatively few combinatorially modified forms dominate the population through Top Down proteomics. The main combinatorial PTMs we find through the Top Down approach are N-terminal acetylation, Arg25 methylation along with phosphorylation of the three most C-terminal serine residues in primarily a diphosphorylated form. This report presents one of the most detailed analyses of HMGA1a to date and illustrates the strength of using a combined MS effort.

Specific interactions between post-translational modifications (PTMs) and chromatin-binding proteins are central to the idea of a 'histone code'. Here, we used a 5,000-member, PTM-randomized, combinatorial peptide library based on the N terminus of histone H3 to interrogate the multisite specificity of six chromatin binding modules, which read the methylation status of Lys4. We found that Thr3 phosphorylation, Arg2 methylation and Thr6 phosphorylation are critical additional PTMs that modulate the ability to recognize and bind histone H3. Notably, phosphorylation of Thr6 yielded the most varied effect on protein binding, suggesting an important regulatory mechanism for readers of the H3 tail. Mass spectrometry and antibody-based evidence indicate that this previously uncharacterized modification exists on native H3, and NMR analysis of ING2 revealed the structural basis for discrimination. These investigations reveal a continuum of binding affinities in which multisite PTM recognition involves both switch- and rheostat-like properties, yielding graded effects that depend on the inherent 'reader' specificity.

Protein post-translational modifications (PTMs) have been widely shown to influence protein-protein interactions, direct subcellular location and transduce a variety of both internal and externally generated signals into cellular/phenotypic outcomes. Mass spectrometry has been a key tool for the elucidation of several types of PTMs in both qualitative and quantitative manners. As large datasets on the proteome-wide level are now being generated on a daily basis, the identification of combinatorial PTM patterns has become feasible. A survey of the recent literature in this area shows that many proteins undergo multiple modifications and that sequential or hierarchal patterns exist on many proteins; the biology of these modification patterns is only starting to be unraveled. This review will outline combinatorial PTM examples in biology, and the mass spectrometry-based techniques and applications utilized in the investigations of these combinatorial PTMs.

Epigenetics is increasingly being recognized as a central component of physiological processes as diverse as obesity and circadian rhythms. Primarily acting through DNA methylation and histone post-translational modifications, epigenetic pathways enable both short- and long-term transcriptional activation and silencing, independently of the underlying genetic sequence. To more quantitatively study the molecular basis of epigenetic regulation in physiological processes, the present review informs the latest techniques to identify and compare novel DNA methylation marks and combinatorial histone modifications across different experimental conditions, and to localize both DNA methylation and histone modifications over specific genomic regions. Key words: mass spectrometry, proteomics, histone, chromatin.

A novel algorithm, PILOT_PTM, has been developed for the untargeted identification of post-translational modifications (PTMs) on a template sequence. The algorithm consists of an analysis of a tandem mass spectrum (MS/MS) via an integer linear optimization model to output a rank-ordered list of PTMs that best match the experimental data. Each MS/MS is analyzed by a preprocessing algorithm to reduce spectral noise and label potential complimentary, offset, isotope, and multiply charged peaks. Postprocessing of the rank-ordered list from the integer linear optimization model will resolve fragment mass errors and will re-order the list of PTMs based on the cross-correlation between the experimental and theoretical MS/MS. PILOT_PTM is instrument independent, capable of handling multiple fragmentation technologies, and can address the universe of PTMs for every amino acid on the template sequence. The various features of PILOT_PTM are presented and it is tested on several modified and unmodified data sets including chemically synthesized phosphopeptides, histone H3 (1-50) polypeptides, histone H3 tryptic fragments, and peptides generated from proteins extracted from chromatin enriched fractions. The data sets consist of spectra derived from fragmentation via collision induced dissociation, electron transfer dissociation, and electron capture dissociation. The capability of PILOT_PTM is then benchmarked using five state-of-the-art methods InsPecT, VEMS, Modi, MASCOT, and X!Tandem. PILOT_PTM demonstrates superior accuracy on both the small and large-scale proteome experiments. A protocol is finally developed for the analysis of a complete LC-MS/MS scan using template sequences generated from SEQUEST and is demonstrated on over 270,000 MS/MS collected from a total chromatin digest.

INTRODUCTION: Studies of type 2 translation, the adaption of evidence-based interventions to real-world settings, should include representative study sites and staff to improve external validity. Sites for such studies are, however, often selected by convenience sampling, which limits generalizability. We used an optimized probability sampling protocol to select an unbiased, representative sample of study sites to prepare for a randomized trial of a weight loss intervention. METHODS: We invited North Carolina health departments within 200 miles of the research center to participate (N = 81). Of the 43 health departments that were eligible, 30 were interested in participating. To select a representative and feasible sample of 6 health departments that met inclusion criteria, we generated all combinations of 6 from the 30 health departments that were eligible and interested. From the subset of combinations that met inclusion criteria, we selected 1 at random. RESULTS: Of 593,775 possible combinations of 6 counties, 15,177 (3%) met inclusion criteria. Sites in the selected subset were similar to all eligible sites in terms of health department characteristics and county demographics. CONCLUSION: Optimized probability sampling improved generalizability by ensuring an unbiased and representative sample of study sites.

Mass spectrometry (MS) research has revolutionized modern biological and biomedical fields. At the heart of the majority of mass spectrometry experiments is the use of Bottom Up mass spectrometry methods where proteins are first proteolyzed into smaller fragments before MS interrogation. The advent of electron capture dissociation and, more recently, electron-transfer dissociation, however, has allowed Top Down (analysis of intact proteins) or middle down (analysis of large polypeptides) mass spectrometry to both experience large increases in development, growth, and overall usage. Nevertheless, for high-throughput large-scale proteomic studies, Bottom Up mass spectrometry has easily dominated the field. As Top Down mass spectrometry methodology and technology continue to develop, will it genuinely be able to compete with Bottom Up mass spectrometry for whole proteome analysis? Discussed here are the current approaches, applications, issues, and future view of high-throughput Top Down mass spectrometry.

Methylation of specific histone residues is capable of both gene activation and silencing. Despite vast work on the function of methylation, most studies either present a static snapshot of methylation or fail to assign kinetic information to specific residues. Using liquid chromatography-tandem mass spectrometry on a high-resolution mass spectrometer and heavy methyl-SILAC labeling, we studied site-specific histone lysine and arginine methylation dynamics. The detection of labeled intermediates within a methylation state revealed that mono-, di-, and trimethylated residues generally have progressively slower rates of formation. Furthermore, methylations associated with active genes have faster rates than methylations associated with silent genes. Finally, the presence of both an active and silencing mark on the same peptide results in a slower rate of methylation than the presence of either mark alone. Here we show that quantitative proteomic approaches such as this can determine the dynamics of multiple methylated residues, an understudied portion of histone biology.

Covalent adenosine diphosphate (ADP)-ribosylation of proteins is a post-translational modification that can occur both enzymatically and nonenzymatically and has been linked to many biological processes, such as the DNA damage and response mechanisms. Although the biochemistry of protein ADP-ribosylation has been extensively studied, the identification of physiological substrates remains a significant challenge due to inadequate tools for characterizing these modified peptides. Here we show that the use of electron transfer dissociation (ETD) greatly simplifies the mass spectrometric (MS) sequencing of ADP-ribosylated peptides over the conventional collisionally activated dissociation (CAD) approach. ETD sequencing of ADP-ribosylated peptides can be performed within a time scale compatible with nanoflow liquid chromatography and should prove useful for high-throughput large-scale studies on this unique class of modified peptides.

Despite increasing applications of mass spectrometry (MS) to characterize post-translational modifications (PTMs) on histone proteins, most existing protocols are not properly suited to robustly measure them in a high-throughput quantitative manner. In this work, we expand on current protocols and describe improved methods for quantitative Bottom Up characterization of histones and their PTMs with comparable sensitivity, but much higher throughput than standard MS approaches. This is accomplished by first bypassing off-line fractionation of histone proteins and working directly with total histones from a typical nuclei acid extraction. Next, using a chemical derivatization procedure that is combined with stable-isotope labeling in a two-step process, we can quantitatively compare samples using nanoLC-MS/MS. We show that our method can successfully detect 17 combined H2A/H2B variants and over 25 combined histone H3 and H4 PTMs in a single MS experiment. We test our method by quantifying differentially expressed histone PTMs from wild-type yeast and a methyltransferase knockout strain. This improved methodology establishes that time and sample consuming off-line HPLC or SDS-PAGE purification of individual histone variants prior to MS interrogation as commonly performed is not strictly required. Our protocol significantly streamlines the analysis of histone PTMs and will allow for studies of differentially expressed PTMs between multiple samples during biologically relevant processes in a rapid and quantitative fashion.