Biomedical subjects

Philip R Gafken

Publications and source records attributed to Philip R Gafken.

10 recordsLinked to original sources

PubMed2006

Ultrasensitive detection and characterization of posttranslational modifications using surface-enhanced Raman spectroscopy.

Posttranslational modification (PTM) of proteins is likely to be the most common mechanism of altering the expression of genetic information. It is essential to characterize PTMs to establish a complete understanding of the activities of proteins. Here, we present a sensitive detection method using surface-enhanced Raman spectroscopy (SERS) that can detect PTMs from as little as zeptomoles of peptide. We demonstrate, using model peptides, the ability of SERS to detect a variety of protein modifications, such as acetylation, trimethylation, phosphorylation, and ubiquitination. In addition, we show the capability to obtain positional information for modifications such as trimethylation and phosphorylation using SERS and wavelet decomposition data analysis techniques. We further show that it is possible to apply SERS to detect PTMs from biological samples such as histones. We envision that this detection method might be a valuable technique that is complementary to mass spectrometry in obtaining orthogonal chemical and modification-specific information from biological samples at sensitive levels.

Acetylation↗

PubMed2006

Myc influences global chromatin structure.

The family of myc proto-oncogenes encodes transcription factors (c-, N-, and L-Myc) that regulate cell growth and proliferation and are involved in the etiology of diverse cancers. Myc proteins are thought to function by binding and regulating specific target genes. Here we report that Myc proteins are required for the widespread maintenance of active chromatin. Disruption of N-myc in neuronal progenitors and other cell types leads to nuclear condensation accompanied by large-scale changes in histone modifications associated with chromatin inactivation, including hypoacetylation and altered methylation. These effects are largely reversed by exogenous Myc as well as by differentiation and are mimicked by the Myc antagonist Mad1. The first chromatin changes are evident within 6 h of Myc loss and lead to changes in chromatin structure. Myc widely influences chromatin in part through upregulation of the histone acetyltransferase GCN5. This study provides the first evidence for regulation of global chromatin structure by an oncoprotein and may explain the broad effects of Myc on cell behavior and tumorigenesis.

Acetylation↗

PubMed2005

Evaluation of matrix-assisted laser desorption/ionization-time of flight mass spectrometry proteomic profiling: identification of alpha 2-HS glycoprotein B-chain as a biomarker of diet.

Biomarkers have the potential to impact a wide range of public health concerns, including early detection of diseases, drug discovery, and improved accuracy of monitoring effects of interventions. Given new technological developments, broad-based screening approaches will likely advance biomarker discovery at an accelerated pace. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) allows for the elucidation of individual protein masses from a complex mixture with high throughput. We have developed a method for identifying serum biomarkers using MALDI-TOF and statistical analysis. However, before applying this approach to screening of complex diseases, we evaluated the approach in a controlled dietary intervention study. In this study, MALDI-TOF spectra were generated using samples from a randomized controlled trial. During separate feeding periods, 38 participants ate a basal diet devoid of fruits and vegetables and a basal diet supplemented with cruciferous (broccoli) family vegetables. Serum samples were obtained at the end of each 7-day feeding period and treated to remove large, abundant proteins. MALDI-TOF spectra were analyzed using peak picking algorithms and logistic regression models. Our bioinformatics methods identified two significant peaks at m/z values of 2740 and 1847 that could classify participants based on diet (basal vs. cruciferous) with 76% accuracy. The 2740 m/z peak was identified as the B-chain of alpha 2-HS glycoprotein, a serum protein previously found to vary with diet and be involved in insulin resistance and immune function.

Adult↗

PubMed2004

Mass spectrometry of UV-cross-linked protein-nucleic acid complexes: identification of amino acid residues in the single-stranded DNA-binding domain of human replication protein A.

Photochemical cross-linking of human replication protein A (hRPA) to oligonucleotide dT30 was performed to enable identification of amino acid sequences that reside in the DNA-binding domain. A nucleoprotein complex, with a 1:1 protein/DNA stoichiometry, was separated from unreacted enzyme and oligonucleotide by SDS-polyacrylamide gel electrophoresis and subjected to in-gel digestion with trypsin. Three cross-linked tryptic peptides (nucleopeptides) of hRPA70xdT30 (T43, T28/29, and a truncated T24/25) were isolated. Combined mass spectrometric and C-terminal proteolysis experiments showed that at least one amino acid in the segment 235-ATAFNE-240 (located in T24/25), at least one out of the two residues sequence 269-FT-270 (located in T28/29), and at least one from the sequence 383-VSDF-386 (located in T43) were involved in cross-linking. These peptides contained aromatic residues (F238, F269, and F386 respectively) that can form base-stacking interactions with the DNA and were, therefore, most likely to be involved in cross-linking. The results obtained in this study demonstrate that a combination of exhaustive proteolysis and MALDI TOF MS can localize the sites of DNA binding to very short sequences of amino acids. Data so acquired can confirm or amend information obtained from site-directed mutagenesis and X-ray crystallography.

Amino Acid Sequence↗

PubMed2004

Histone H3.3 is enriched in covalent modifications associated with active chromatin.

Chromatin states can be distinguished by differential covalent modifications of histones or by utilization of histone variants. Chromatin associated with transcriptionally active loci becomes enriched for histones with particular lysine modifications and accumulates the H3.3 histone variant, the substrate for replication-independent nucleosome assembly. However, studies of modifications at particular loci have not distinguished between histone variants, so the relationship among modifications, histone variants, and nucleosome assembly pathways is unclear. To address this uncertainty, we have quantified the relative abundance of H3 and H3.3 and their lysine modifications. Using a Drosophila cell line system in which H3.3 has been shown to specifically package active loci, we found that H3.3 accounts for approximately 25% of total histone 3 in bulk chromatin, enough to package essentially all actively transcribed genes. MS and antibody characterization of separated histone 3 fractions revealed that H3.3 is relatively enriched in modifications associated with transcriptional activity and deficient in dimethyl lysine-9, which is abundant in heterochromatin. To explain enrichment on alternative variants, we propose that histone modifications are tied to the alternative nucleosome assembly pathways that use primarily H3 at replication forks and H3.3 at actively transcribed genes in a replication-independent manner.

Animals↗

PubMed2004

Proteomic analysis of native metabotropic glutamate receptor 5 protein complexes reveals novel molecular constituents.

We used a proteomic approach to identify novel proteins that may regulate metabotropic glutamate receptor 5 (mGluR5) responses by direct or indirect protein interactions. This approach does not rely on the heterologous expression of proteins and offers the advantage of identifying protein interactions in a native environment. The mGluR5 protein was immunoprecipitated from rat brain lysates; co-immunoprecipitating proteins were analyzed by mass spectrometry and identified peptides were matched to protein databases to determine the correlating parent proteins. This proteomic approach revealed the interaction of mGluR5 with known regulatory proteins, as well as novel proteins that reflect previously unidentified molecular constituents of the mGluR5-signaling complex. Immunoblot analysis confirmed the interaction of high confidence proteins, such as phosphofurin acidic cluster sorting protein 1, microtubule-associated protein 2a and dynamin 1, as mGluR5-interacting proteins. These studies show that a proteomic approach can be used to identify candidate interacting proteins. This approach may be particularly useful for neurobiology applications where distinct protein interactions within a signaling complex can dramatically alter the outcome of the response to neurotransmitter release, or the disruption of normal protein interactions can lead to severe neurological and psychiatric disorders.

Algorithms↗

PubMed2003

Mass spectrometric quantification of acetylation at specific lysines within the amino-terminal tail of histone H4.

Electrospray ionization mass spectrometry, a leading method for the quantification of biomolecules, is useful for the analysis of posttranslational modifications of proteins. Here we describe a mass spectrometric approach for determining levels of acetylation at individual lysine residues within the amino-terminal tail of histone H4. Because of the high density of acetylatable lysine residues within this short span of amino acids, collision-induced dissociation tandem mass spectrometry was required. In addition, it was necessary to develop an algorithm to determine the fraction of acetylation at specific lysine residues from fragment ions containing more than one lysine residue. This is the first report of direct measurement of endogeneous levels of acetylation at individual lysine residues within the amino-terminal tail of yeast histone H4 and is the first use of tandem mass spectrometry for quantification of peptides containing multiple sites of modification.

Acetylation↗

PubMed2002

Heritable chromatin structure: mapping "memory" in histones H3 and H4.

Telomeric position effect in Saccharomyces cerevisiae is a chromatin-mediated phenomenon in which telomere proximal genes are repressed (silenced) in a heritable, but reversible, fashion. Once a transcriptional state (active or silenced) is established, however, there is a strong tendency for that state to be propagated. Twenty-five years ago, H. Weintraub and colleagues suggested that such heritability could be mediated by posttranslational modification of chromatin [Weintraub, H., Flint, S. J., Leffak, I. M., Groudine, M. & Grainger, R. M. (1977) Cold Spring Harbor Symp. Quant. Biol. 42, 401-407]. To identify potential sites within the chromatin that might act as sources of "memory" for the heritable transmission, we performed a genetic screen to isolate mutant alleles of the histones H3 and H4 genes that would "lock" telomeric marker genes into a silenced state. We identified mutations in the NH(2)-terminal tail and core of both histones; most of the amino acid changes mapped adjacent to lysines that are known sites of acetylation or methylation. We developed a method using MS to quantify the level of acetylation at each lysine within the histone H4 NH(2)-terminal tail in these mutants. We discovered that each of these mutants had a dramatic reduction in the level of acetylation at lysine 12 within the histone H4 tail. We propose that this lysine serves as a "memory mark" for propagating the expression state of a telomeric gene: when it is unacetylated, silent chromatin will be inherited; when it is acetylated an active state will be inherited.

Acetylation↗

PubMed2002

Dot1p modulates silencing in yeast by methylation of the nucleosome core.

DOT1 was originally identified as a gene affecting telomeric silencing in S. cerevisiae. We now find that Dot1p methylates histone H3 on lysine 79, which maps to the top and bottom of the nucleosome core. Methylation occurs only when histone H3 is assembled in chromatin. In vivo, Dot1p is solely responsible for this methylation and methylates approximately 90% of histone H3. In dot1delta cells, silencing is compromised and silencing proteins become redistributed at the expense of normally silenced loci. We suggest that methylation of histone H3 lysine 79 limits silencing to discrete loci by preventing the binding of Sir proteins elsewhere along the genome. Because Dot1p and histone H3 are conserved, similar mechanisms are likely at work in other eukaryotes.

Amino Acid Motifs↗

PubMedDate not supplied

Methodologies for characterizing phosphoproteins by mass spectrometry.

Posttranslational regulation of proteins via protein phosphorylation is one of the major means of protein regulation. Phosphorylation is a very rapid and reversible method of changing the function of proteins. Detection of phosphorylated proteins and the identification of phosphorylation sites are necessary to molecularly link specific phosphorylated events with change in phosphoprotein function. Mass Spectrometry (MS) has become the methodology of choice for phosphosite identification. Here we review current approaches including sample separation and enrichment techniques (SDS-PAGE, immunoprecipitation, metal-assisted enrichment, strong cation exchange, dendrimer capture), quantitative MS analysis methods (SILAC, iTRAQ, AQUA), and the application of recently developed methods including electron transfer dissociation ionization and "top-down" proteomics to phosphoprotein analysis.

Mass Spectrometry↗