Search

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. 10.1021/ac902134y

A combination of immobilized metal affinity chromatography and tandem mass spectrometry is used to characterize post-translational modifications associated with phosphorylation on the N-terminal tails of histone H3 variants purified from mitotically arrested HeLa cells. The methods provide a powerful approach for elucidation of the histone code and identification of histone post-translational modifications that occur during mitosis and other specific cellular events.

A new method of sample preparation was developed for MALDI-TOF-MS analysis of oligonucleotides. First, aqueous DNA samples are dispensed and allowed to dry. Then 6-aza-2-thiothymine matrix dissolved in nonaqueous volatile solvents is applied on top of the DNA residue to form a thin homogeneous film. MALDI-TOF analysis shows such preparation generates much better shot-to-shot and sample-to-sample reproducibility and essentially eliminates the need to search for 'hot' spots. The increased homogeneity of the matrix/analyte crystal distribution results in significant improvement for quantitative and high-throughput analyses of DNA. Using this method, isotopically resolved oligonucleotide spectra up to a 24-mer can also be easily obtained in a reflectron instrument. Due to the ease of preparation, this method could be widely useful for a number of applications such as for assays that are performed on surface in vitro, as the thin-layer matrix could be applied directly for MALDI analysis.

The apparent mass resolution of oligonucleotides in time-of-flight (TOF) mass spectrometers has been examined. In a reflectron TOF instrument, where the isotopic profile can be completely resolved, the apparent resolution matches the instrument's resolving power. In a linear TOF instrument, unresolved isotopic profiles limit the apparent resolution to much lower values than the actual instrument resolution. By using [sup.12]C/[sup.14]N-enriched oligonucleotides, the apparent resolution can be improved significantly. The isotope enrichment method also enhances the signal-to-noise ratio.

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. [Copyright &y& Elsevier]

RNA is dynamically modified in cells by a plethora of chemical moieties to modulate molecular functions and processes. Over 140 modifications have been identified across species and RNA types, with the highest density and diversity of modifications found in tRNA (tRNA). The methods used to identify and quantify these modifications have developed over recent years and continue to advance, primarily in the fields of next-generation sequencing (NGS) and mass spectrometry (MS). Most current NGS methods are limited to antibody-recognized or chemically derivatized modifications and have limitations in identifying multiple modifications simultaneously. Mass spectrometry can overcome both of these issues, accurately identifying a large number of modifications in a single run. Here, we present advances in MS data acquisition for the purpose of RNA modification identification and quantitation. Using this approach, we identified multiple tRNA wobble position modifications in Arabidopsis thaliana that are upregulated in salt-stressed growth conditions and may stabilize translation of salt stress induced proteins. This work presents improvements in methods for studying RNA modifications and introduces a possible regulatory role of wobble position modifications in A. thaliana translation. [ABSTRACT FROM AUTHOR]

Examines the mass resolution of oligonucleotides in time-of-flight (TOF) mass spectrometers. Compatibility of the apparent resolution with the resolving power of the instrument; Limitation of the isotopic profiles in a linear TOF instrument; Improvement of the apparent resolution by using [sup 12]C/[sup 14]N-enriched oligonucleotides.

The cell cycle is a highly regulated and evolutionary conserved process that results in the duplication of cell content and the equal distribution of the duplicated chromosomes into a pair of daughter cells. Histones are fundamental structural components of chromatin in eukaryotic cells, and their post-translational modifications (PTMs) benchmark DNA readout and chromosome condensation. Aberrant regulation of the cell cycle associated with dysregulation of histone PTMs is the cause of critical diseases such as cancer. Monitoring changes of histone PTMs could pave the way to understanding the molecular mechanisms associated with epigenetic regulation of cell proliferation. Previously, our lab established a novel middle-down workflow using porous graphitic carbon (PGC) as a stationary phase to analyze histone PTMs, which utilizes the same reversed-phase chromatography for gradient separation as canonical proteomics coupled with online mass spectrometry (MS). Here, we applied this novel workflow for high-throughput analysis of histone modifications of H3.1 and H3.2 during the cell cycle. Collectively, we identified 1133 uniquely modified canonical histone H3 N-terminal tails. Consistent with previous findings, histone H3 phosphorylation increased significantly during the mitosis (M) phase. Histone H3 variant-specific and cell-cycle-dependent expressions of PTMs were observed, underlining the need to not combine H3.1 and H3.2 together as H3. We confirmed previously known H3 PTM crosstalk (e.g. K9me–S10ph) and revealed new information in this area as well. These findings imply that the combinatorial PTMs play a role in cell cycle control, and they may serve as markers for proliferation. [ABSTRACT FROM AUTHOR]

The analysis of histone post-translational modifications (PTMs) by mass spectrometry (MS) has been critical to the advancement of the field of epigenetics. The most sensitive and accurate workflow is similar to the canonical proteomics analysis workflow (bottom-up MS), where histones are digested into short peptides (4-20 aa) and quantitated in extracted ion chromatograms. However, this limits the ability to detect even very common co-occurrences of modifications on histone proteins, preventing biological interpretation of PTM crosstalk. By digesting with GluC rather than trypsin, it is possible to produce long polypeptides corresponding to intact histone N-terminal tails (50-60 aa), where most modifications reside. This middle-down MS approach is used to study distant PTM co-existence. However, the most sensitive middle-down workflow uses weak cation exchange-hydrophilic interaction chromatography (WCX-HILIC), which is less robust than conventional reversed-phase chromatography. Additionally, since the buffer systems for middle-down and bottom-up proteomics differ substantially, it is cumbersome to toggle back and forth between both experimental setups on the same LC system. Here, we present a new workflow using porous graphitic carbon (PGC) as a stationary phase for histone analysis where bottom-up and middle-down sized histone peptides can be analyzed simultaneously using the same reversed-phase buffer setup. By using this protocol for middle-down sized peptides, we identified 406 uniquely modified intact histone tails and achieved a correlation of 0.85 between PGC and WCX-HILIC LC methods. Together, our method facilitates the analysis of single and combinatorial histone PTMs with much simpler applicability for conventional proteomics labs than the state-of-the-art middle-down MS. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

The eukaryotic genome is packaged around histone proteins, which are subject to a myriad of post-translational modifications. By controlling DNA accessibility and the recruitment of protein complexes that mediate chromatin-related processes, these modifications constitute a key mechanism of epigenetic regulation. Since mass spectrometry can easily distinguish between these different modifications, it has become an essential technique in deciphering the histone code. Although robust LC-MS/MS methods are available to analyze modifications on the histone N-terminal tails, routine methods for characterizing ubiquitin marks on histone C-terminal regions, especially H2AK119ub, are less robust. Here, we report the development of a simple workflow for the detection and improved quantification of the canonical histone ubiquitination marks H2AK119ub and H2BK120ub. The method entails a fully tryptic digestion of acid-extracted histones, followed by derivatization with heavy or light propionic anhydride. A pooled sample is then spiked into oppositely labeled single samples as a reference channel for relative quantification, and data is acquired using PRM-based nano-LC-MS/MS. We validated our approach with synthetic peptides as well as treatments known to modulate the levels of H2AK119ub and H2BK120ub. This new method complements existing histone workflows, largely focused on the lysine-rich N-terminal regions, by extending modification analysis to other sequence contexts.

We set out to determine whether the C-terminus (amino acids 481-798) of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α, UniProt Q9UBK2), a regulatory metabolic protein involved in mitochondrial biogenesis, and respiration, is an arginine methyltransferase substrate. Arginine methylation by protein arginine methyltransferases (PRMTs) alters protein function and thus contributes to various cellular processes. In addition to confirming methylation of the C-terminus by PRMT1 as described in the literature, we have identified methylation by another member of the PRMT family, PRMT7. We performed in vitro methylation reactions using recombinant mammalian PRMT7 and PRMT1 at 37, 30, 21, 18, and 4 °C. Various fragments of PGC-1α corresponding to the C-terminus were used as substrates, and the methylation reactions were analyzed by fluorography and mass spectrometry to determine the extent of methylation throughout the substrates, the location of the methylated PGC-1α arginine residues, and finally, whether temperature affects the deposition of methyl groups. We also employed two prediction programs, PRmePRed and MePred-RF, to search for putative methyltransferase sites. Methylation reactions show that arginine residues R548 and R753 in PGC-1α are methylated at or below 30 °C by PRMT7, while methylation by PRMT1 was detected at these same residues at 30 °C. Computational approaches yielded additional putative methylarginine sites, indicating that since PGC-1α is an intrinsically disordered protein, additional methylated arginine residues have yet to be experimentally verified. We conclude that temperature affects the extent of arginine methylation, with more methylation by PRMT7 occurring below physiological temperature, uncovering an additional control point for PGC-1α.

Massive formalin-fixed, paraffin-embedded (FFPE) tissue archives exist worldwide, representing an invaluable resource for clinical proteomics research. However, current protocols for FFPE proteomics lack standardization, efficiency, reproducibility, and scalability. Here we present high-yield protein extraction and recovery by direct solubilization (HYPERsol), an optimized workflow using ultrasonication and S-Trap sample processing that enables proteome coverage and quantification from FFPE samples comparable to that achieved from flash-frozen tissue (average R = 0.936). When applied to archival samples, HYPERsol resulted in high-quality data from FFPE specimens in storage for up to 17 years, and may enable the discovery of new immunohistochemical markers.

Histone post-translational modifications (PTMs) are fundamental players of chromatin regulation, as they contribute to editing histone chemical properties and recruiting proteins for gene transcription and DNA repair. Mass spectrometry (MS)-based proteomics is currently the most widely adopted strategy for high-throughput quantification of hundreds of histone PTMs. Samples such as primary tissues, complex model systems, and biofluids are hard to retrieve in large quantities. Because of this, it is critical to know whether the amount of sample available would lead to an exhaustive analysis if subjected to MS. In this work, we assessed the reproducibility in quantification of histone PTMs using a wide range of starting material, that is, from 5 000 000 to 50 000 cells. We performed the experiment using four different cell lines, that is, HeLa, 293T, human embryonic stem cells (hESCs), and myoblasts, and we quantified a list of 205 histone peptides using ion trap MS and our in-house software. Results highlighted that the relative abundance of some histone PTMs deviated as little as just 4% when comparing high starting material with histone samples extracted from 50 000 cells, for example, H3K9me2 (40% average abundance). Low abundance PTMs such as H3K4me2 (<3% average abundance) showed higher variability, but still ∼34%. This indicates that most PTMs, and especially abundant ones, are quantified with high precision starting from low cell counts. This study will help scientists to decide whether specific experiments are feasible and to plan how much sample should be reserved for histone analysis using MS.