RESUMEN
A novel protein identification framework, PILOT_PROTEIN, has been developed to construct a comprehensive list of all unmodified proteins that are present in a living sample. It uses the peptide identification results from the PILOT_SEQUEL algorithm to initially determine all unmodified proteins within the sample. Using a rigorous biclustering approach that groups incorrect peptide sequences with other homologous sequences, the number of false positives reported is minimized. A sequence tag procedure is then incorporated along with the untargeted PTM identification algorithm, PILOT_PTM, to determine a list of all modification types and sites for each protein. The unmodified protein identification algorithm, PILOT_PROTEIN, is compared to the methods SEQUEST, InsPecT, X!Tandem, VEMS, and ProteinProspector using both prepared protein samples and a more complex chromatin digest. The algorithm demonstrates superior protein identification accuracy with a lower false positive rate. All materials are freely available to the scientific community at http://pumpd.princeton.edu.
Asunto(s)
Algoritmos , Proteínas/química , Análisis de Secuencia de Proteína/métodos , Programas Informáticos , Espectrometría de Masas en Tándem , Análisis por Conglomerados , Bases de Datos de Proteínas , Fragmentos de Péptidos/química , Procesamiento Proteico-Postraduccional , Proteómica , Curva ROCRESUMEN
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 an MS/MS spectrum via an integer linear optimization model to output a rank-ordered list of PTMs that best match the experimental data. Each MS/MS spectrum 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 reorder the list of PTMs based on the cross-correlation between the experimental and theoretical MS/MS spectrum. 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-(1-50) 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, Virtual Expert Mass Spectrometrist (VEMS), Mod(i), 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 spectra collected from a total chromatin digest.
Asunto(s)
Algoritmos , Procesamiento Proteico-Postraduccional , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Animales , Cromatografía Liquida , Bases de Datos de Proteínas , Células HeLa , Histonas/química , Histonas/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Fosfopéptidos/síntesis química , Fosfopéptidos/química , Reproducibilidad de los ResultadosRESUMEN
Neuropsychiatric disorders affect a large segment of the human population and result in large costs to society. The majority of such disorders have unknown underlying causes. Recent evidence suggests an important role for epigenetic regulation in the emergence of neuropsychiatric disease. Epigenetics may provide a link between genetic and environmental factors and behavior. Epigenetic signaling involves changes on the structure of chromatin; such changes are often triggered and maintained by the post-translational modification of chromatin proteins and/or DNA. Recent proteomic technologies have enabled the study of epigenetic mechanisms in a high-throughput manner. This review will provide an overview of the major epigenetic pathways and modern techniques for their study, before focusing on experimental evidence supporting a strong role for epigenetics in selected psychiatric disorders such as depression, schizophrenia, and drug addiction. These results highlight a great need for the inclusion of the proteomic characterization of epigenetic mechanisms in the study of gene/disease associations in psychiatric disorders.
Asunto(s)
Epigenómica/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Trastornos Mentales , Proteómica/métodos , Cromatina/química , Cromatina/metabolismo , Metilación de ADN , Depresión , Trastornos Mentales/diagnóstico , Trastornos Mentales/tratamiento farmacológico , Trastornos Mentales/metabolismo , Trastornos Mentales/fisiopatología , Esquizofrenia , Transducción de Señal , Trastornos Relacionados con SustanciasRESUMEN
Heterochromatin protein 1 (HP1) family members (alpha, beta, and gamma) bind histone H3 methylated at Lys-9, leading to gene silencing and heterochromatin formation. Several previous reports have suggested that HP1s are post-translationally modified, yet sites of modification have not yet been exhaustively determined. Here we perform the first comprehensive proteomic analysis of all HP1 isoforms using tandem mass spectrometry. Our data reveal that all HP1 isoforms are highly modified in a manner analogous to histones including phosphorylation, acetylation, methylation, and formylation, including several sites having multiple different types of modifications. Additionally, many of these modifications are found in both the chromo- and chromoshadow domains, suggesting that they may have an important role in modulating HP1 interactions or functions. These studies are the first to systematically map the abundant sites of covalent modifications on HP1 isoforms and provide the foundation for future investigations to test whether these modifications are essential in heterochromatin maintenance or other nuclear processes.
Asunto(s)
Proteínas Cromosómicas no Histona/química , Histonas/química , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Núcleo Celular/metabolismo , Cromatografía Liquida/métodos , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Drosophila , Humanos , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Péptidos/química , Isoformas de Proteínas , Proteómica/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
We present a novel method utilizing "saltless" pH gradient weak cation exchange-hydrophilic interaction liquid chromatography directly coupled to electron transfer dissociation (ETD) mass spectrometry for the automated on-line high throughput characterization of hypermodified combinatorial histone codes. This technique, performed on a low resolution mass spectrometer, displays an improvement over existing methods with an approximately 100-fold reduction in sample requirements and analysis time. The scheme presented is capable of identifying all of the major combinatorial histone codes present in a sample in a 2-h analysis. The large N-terminal histone peptides are eluted by the pH and organic solvent weak cation exchange-hydrophilic interaction liquid chromatography gradient and directly introduced via nanoelectrospray ionization into a benchtop linear quadrupole ion trap mass spectrometer equipped with ETD. Each polypeptide is sequenced, and the modification sites are identified by ETD fragmentation. The isobaric trimethyl and acetyl modifications are resolved chromatographically and confidently distinguished by the synthesis of mass spectrometric and chromatographic information. We demonstrate the utility of the method by complete characterization of human histone H3.2 and histone H4 from butyrate-treated cells, but it is generally applicable to the analysis of highly modified peptides. We find this methodology very useful for chromatographic separation of isomeric species that cannot be separated well by any other chromatographic means, leading to less complicated tandem mass spectra. The improved separation and increased sensitivity generated novel information about much less abundant forms. In this method demonstration we report over 200 H3.2 forms and 70 H4 forms, including forms not yet detected in human cells, such as the remarkably highly modified histone H3.2 K4me3K9acK14acK18acK23acK27acK36me3. Such detail provided by our proteomics platform will be essential for determining how histone modifications occur and act in combination to propagate the histone code during transcriptional events and could greatly enable sequencing of the histone component of human epigenomes.
Asunto(s)
Cromatografía Liquida/métodos , Código de Histonas , Espectrometría de Masas/métodos , Secuencia de Aminoácidos , Butiratos/química , Células HeLa , Histonas/química , Histonas/genética , Humanos , Isomerismo , Datos de Secuencia Molecular , Péptidos/análisisRESUMEN
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.
Asunto(s)
Procesamiento Proteico-Postraduccional , Proteínas/análisis , Proteómica/métodos , Espectrometría de Masas/métodosRESUMEN
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.
Asunto(s)
Histonas/química , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Algoritmos , Cromatografía Liquida/métodos , Proteínas Fúngicas/química , Células HeLa , Humanos , Flujo de TrabajoRESUMEN
Histones are highly conserved proteins that organize cellular DNA. These proteins, especially their N-terminal domains, are adorned with many post-translational modifications (PTMs) such as lysine methylation, which are associated with active or repressed transcriptional states. The lysine methyltransferase G9a and its interaction partner Glp1 can mono- or dimethylate histone H3 on lysine (H3K9me1 or me2); possible cross-talk between these modifications and other PTMs on the same or other histone molecules is currently uncharacterized. In this study, we comprehensively analyze the effects of G9a/Glp1 knockdown on the most abundant histone modifications through both Bottom Up and Middle Down mass spectrometry-based proteomics. In addition to the expected decrease in H3K9me1/me2 we find that other degrees of methylation on K9 are affected by the reduction of G9a/Glp1 activity, particularly when K9 methylation occurs in combination with K14 acetylation. In line with this, an increase in K14 acetylation upon G9a knockdown was observed across all H3 variants (H3.1, H3.2 and H3.3), hinting at the potential existence of a binary switch between K9 methylation and K14 acetylation. Interestingly, we also detect changes in the abundance of other modifications (such as H3K79me2) in response to lowered levels of G9a/Glp1 suggesting histone PTM cross-talk amongst the H3 variants. In contrast, we find that G9a/Glp1 knockdown produces little effect on the levels of histone H4 PTMs, indicating low to no trans-histone PTM crosstalk. Lastly, we determined gene expression profiles of control and G9a/Glp1 knockdown cells, and we find that the G9a/Glp1 knockdown influences several genes, including DNA binding proteins and key factors in chromatin. Our results provide new insights into the intra- and inter- histone cross-regulation of histone K9 methylation and its potential downstream gene targets.
Asunto(s)
Código de Histonas/genética , Metiltransferasas/genética , Proteómica/métodos , Línea Celular , Cromatografía Líquida de Alta Presión , Humanos , Espectrometría de Masas , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
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.