Biomedical subjects

A L Burlingame

Publications and source records attributed to A L Burlingame.

At least 19 recordsLinked to original sources

PubMed2006

Side-chain fragmentation of alkylated cysteine residues in electron capture dissociation mass spectrometry.

The recent development of novel fragmentation processes based on either electron capture directly or transfer from an anion show great potential for solving problems in proteomics that are intractable by the more widely employed thermal-based fragmentation processes such as collision induced dissociation. The dominant fragmentation occurring upon electron capture dissociation of peptides is cleavage of N-C alpha bonds in the peptide backbone to form c and z* ions. In the case of disulfide-linked peptides, it has also been shown that electron capture on one of the cystine sulfur atoms is favored, resulting in cleavage of the disulfide bond. In this study, we report that electron capture on the sulfur of alkylated cysteine residues is also a dominant process, causing cysteine side-chain loss from z* ions.

Alkylation↗

PubMed2006

Electrospray mass spectrometry of NeuAc oligomers associated with the C fragment of the tetanus toxin.

The Clostridial neurotoxins, botulinum and tetanus, gain entry into motor neurons by binding to the sialic or N-acetylneuraminic acid (NeuAc) residues of gangliosides and specific protein receptors attached to the cell's surface. While the C-fragment of tetanus toxin (TetC) has been identified to be the ganglioside binding domain, remarkably little is known about how this domain discriminates between the structural features of different gangliosides. We have used electrospray ionization mass spectrometry (ESI-MS) to examine the formation of complexes between TetC and carbohydrates containing NeuAc groups to determine how NeuAc residues contribute to ganglioside binding. ESI-MS was used to obtain an estimate of the dissociation constants (KD values) for TetC binding to a number of related NeuAc-containing carbohydrates (sialyllactose and disialyllactose), as well as six (NeuAc)n oligomers (n = 1-6). KD values were found to range between approximately 10-35 microM. The strength of the interactions between the C fragment and (NeuAc)n are consistent with the topography of the targeting domain of tetanus toxin and the nature of its carbohydrate binding sites. These results suggest that the targeting domain of tetanus toxin contains two binding sites that can accommodate NeuAc (or a dimer) and that NeuAc may play an important role in ganglioside binding and molecular recognition, a process critical for normal cell function and one frequently exploited by toxins, bacteria, and viruses to facilitate their entrance into cells.

Binding Sites↗

PubMed2006

Histone chaperone Asf1 is required for histone H3 lysine 56 acetylation, a modification associated with S phase in mitosis and meiosis.

Histone acetylation affects many nuclear processes including transcription, chromatin assembly, and DNA damage repair. Acetylation of histone H3 lysine 56 (H3 K56ac) in budding yeast occurs during mitotic S phase and persists during DNA damage repair. Here, we show that H3 K56ac is also present during premeiotic S phase and is conserved in fission yeast. Furthermore, the H3 K56ac modification is not observed in the absence of the histone chaperone Asf1. asf1delta and H3 K56R mutants exhibit similar sensitivity to DNA damaging agents. Mutational analysis of Asf1 demonstrates that DNA damage sensitivity correlates with (i) decreased levels of H3 K56ac and (ii) a region implicated in histone binding. In contrast, multiple asf1 mutants that are resistant to DNA damage display WT levels of K56ac. These data suggest that maintenance of H3 K56 acetylation is a primary contribution of Asf1 to genome stability in yeast.

Acetylation↗

PubMed2005

Mass spectrometric analysis of histone posttranslational modifications.

The uses of tandem and Fourier transform mass spectrometric methodologies for assignment of the posttranslational sites and occupancies of histones and their isoforms is described employing several illustrative examples. A comparison of information that can be obtained from intact protein sequencing and proteolytic digestion is presented.

Acetylation↗

PubMed2005

Comprehensive mass spectrometric analysis of the 20S proteasome complex.

The 20S proteasome is a multicatalytic protein complex that plays an important role in intracellular protein degradation from archaebacteria to eukaryotes. This complex is made up of two copies each of seven different alpha (alpha) and seven different beta (beta) subunits arranged into four stacked rings (alpha7beta7beta7alpha7). Although the proteasome's cylindrical structure is conserved, the subunit composition of the 20S protein complex varies during the evolution, and the number of subunits increases from archaebacteria to mammals. To fully characterize the 20S proteasome subunit composition and understand the subunit functions, we, the authors of this chapter, have developed and employed various mass spectrometry (MS)-based approaches to generate a comprehensive profile of the 20S proteasomes from rat liver and Tropanosoma brucei. We have identified 7 alpha and 10 beta subunits, including 7 essential and 3 nonessential beta subunits from rat 20S proteasome complex using two-dimensional (2-D) gel electrophoresis and tandem MS (MS/MS). In addition, multiple isoforms of most of the subunits were determined; indicating the composition of rat 20S proteasome complex was much more complicated than expected. Further analysis of the intact protein molecular weight of each subunit using LC-MS confirmed the heterogeneous population of the 20S proteasome and revealed that many of the experimental measured molecular weights do not correspond well with the theoretical values deduced from the sequences in protein databases. This finding is mostly due to the sequence errors in the protein databases and possible posttranslational modifications. Although the protein sequences of rat 20S proteasome are present in the databases, the sequences of the 20S proteasome from T. brucei were not available at the time when the analysis was carried out. To determine the subunit composition of the 20S proteasome from T. brucei, we developed a homology-based database searching tool to identify unknown proteins based on the novel sequences determined by de novo sequencing using MS/MS. As a result, 14 subunits (7 alpha and 7 beta) were identified on the 2-D gel, which was later confirmed by the full-length sequences. Using the same approach, we also identified and characterized an activator protein, PA26, from T. brucei. The purified recombinant PA26 self-assembles into a heptamer ring, which can bind and activate the 20S proteasome from T. brucei as well as rat. Compared to the human PA28 complex, PA26 may be the prototype activator protein involved in proteasomal protein degradation. Therefore, the MS-based strategy developed here for identification of the known and unknown protein complexes can be generalized for the study of other protein complexes.

Amino Acid Sequence↗

PubMed2005

Phosphorylation state of postsynaptic density proteins.

The postsynaptic density (PSD) is an electron-dense structure located at the synaptic contacts between neurons. Its considerable complexity includes cytoskeletal and scaffold proteins, receptors, ion channels and signaling molecules, in line with the role of PSDs in signal transduction and processing. The phosphorylation state of components of the PSD is central to synaptic transmission and is known to play a role in synaptic plasticity, learning and memory. The presence of a range of kinases and phosphatases in the PSD defines potential key players in this context. However, the substrates that these enzymes target have not been fully identified to date. We analyzed the protein composition of purified PSD samples from adult mouse brains by strong cation exchange chromatography fractionation of a tryptic digest followed by nano-reverse phase liquid chromatography coupled with electrospray ionization-quadrupole time of flight tandem mass spectrometry. This led to the identification of 244 proteins. To gain an insight into the phosphoproteome of the PSD we then purified phosphorylated tryptic peptides by immobilized metal ion affinity chromatography. This approach for the specific enrichment of phosphopeptides resulted in the identification of 42 phosphoproteins in the PSD preparation, 39 of which are known PSD components. Here we present a total of 83 in vivo phosphorylation sites.

Animals↗

PubMed2004

Characterization of Tetrahymena histone H2B variants and posttranslational populations by electron capture dissociation (ECD) Fourier transform ion cyclotron mass spectrometry (FT-ICR MS).

This work describes the nature and sequence information content of the electron capture dissociation mass spectra for the intact Tetrahymena histone H2B. Two major variants of this protein were present bearing nominal modifications of both +42 and +84 Da. This work describes identification of the nature of these two modifications. For example, using gas-phase selection and isolation of the +42-Da modified species, from a background of two H2B variants each present in six or more posttranslationally modified isoforms, we were able to determine that this +42-Da modification isoform bears trimethylation rather than acetylation. LC-CIDMS analysis was also employed on digested preparations to obtain complementary detail of the nature of site-specific posttranslational modifications. This study establishes that integration of the information from these two datasets provides a comprehensive map of posttranslational occupancy for each particular covalent assemblage selected for structural investigation.

Amino Acid Sequence↗

PubMed2004

O-sulfonation of serine and threonine: mass spectrometric detection and characterization of a new posttranslational modification in diverse proteins throughout the eukaryotes.

Protein sulfonation on serine and threonine residues is described for the first time. This post-translational modification is shown to occur in proteins isolated from organisms representing a broad span of eukaryote evolution, including the invertebrate mollusk Lymnaea stagnalis, the unicellular malaria parasite Plasmodium falciparum, and humans. Detection and structural characterization of this novel post-translational modification was carried out using liquid chromatography coupled to electrospray tandem mass spectrometry on proteins including a neuronal intermediate filament and a myosin light chain from the snail, a cathepsin-C-like enzyme from the parasite, and the cytoplasmic domain of the human orphan receptor tyrosine kinase Ror-2. These findings suggest that sulfonation of serine and threonine may be involved in multiple functions including protein assembly and signal transduction.

Animals↗

PubMed2004

Spatio-temporal dynamics of the egg-laying-inducing peptides during an egg-laying cycle: a semiquantitative matrix-assisted laser desorption/ionization mass spectrometry approach.

The activity-dependent release of peptides from the neuro-endocrine caudodorsal cell (CDC) system of the freshwater snail Lymnaea stagnalis regulates egg laying and related behaviors. In this study, we optimized a mass spectrometry-based approach to study the spatio-temporal dynamics of peptides that are largely derived from the CDC hormone precursor during an egg-laying cycle and a CDC discharge in vitro. Semi-quantitative peptide mass profiling using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) indicated a massive depletion of peptides from the neurohemal area in the cerebral commissure (COM) during egg laying and the existence of a reserve pool of peptides in the CDC somata that were transported to the COM to restore peptide levels. The depletion of CDC peptides from the COM was correlated to their release during an induced electrical discharge in vitro. Moreover, MALDI-MS of the releasate revealed extensive truncation of the carboxyl terminal peptide. Finally, two novel peptides of 1788 and 5895 Da, not encoded by the CDC hormone precursor, also exhibited temporal quantitative changes similar to those of CDC peptides. Sequencing of the peptide of 1788 Da by tandem mass spectrometry yielded the novel sequence HF(FH)FYGPYDVFQRDVamide. Together, this implicates a more complex set of CDC peptides for the regulation of egg laying than previously anticipated.

Amino Acid Sequence↗

PubMed2003

Regulated expression of apolipoprotein E by human retinal pigment epithelial cells.

In early age-related macular degeneration (AMD), lipid-containing deposits (drusen) accumulate in Bruch's membrane underlying the retinal pigment epithelium (RPE). Recent studies indicate that apolipoprotein E (apoE) may play a role in lipid trafficking in AMD. Compared with the apoE3 allele, the apoE4 and apoE2 alleles are associated with decreased and increased risk for AMD, respectively; drusen contain high levels of apoE, and apoE null mice develop lipid deposits in Bruch's membrane similar to those observed in AMD. Primary cultures of human RPE cells expressing the apoE3 allele were grown on Transwell culture plates. Western blotting, ELISA assay, and mass spectrometry confirmed that apoE3 was secreted into the apical and basal chambers and that secretion was upregulated by thyroid hormone, 9-cis-retinoic acid, and 22(R)-hydroxycholesterol. In addition, basally secreted apoE associated with exogenously added HDL. These results indicate that apoE secretion can be regulated by specific hormones and that apoE associates with HDL. The findings are consistent with a role for apoE in lipid trafficking through Bruch's membrane and may be relevant to AMD.

Adult↗

PubMed2003

Mass spectrometric analysis of protein mixtures at low levels using cleavable 13C-isotope-coded affinity tag and multidimensional chromatography.

In order to identify and compare the protein content of very low quantity samples of high complexity, a protocol has been established that combines the differential profiling strength of a new cleavable 13C isotope-coded affinity tag (cICAT) reagent with the high sequence coverage provided by multidimensional liquid chromatography and two modes of tandem mass spectrometry. Major objectives during protocol optimization were to minimize sample losses and establish a robust procedure that employs volatile buffer systems that are highly compatible with mass spectrometry. Cleavable ICAT-labeled tryptic peptides were separated from nonlabeled peptides by avidin affinity chromatography. Subsequently, peptide samples were analyzed by nanoflow liquid chromatography electrospray ionization tandem mass spectrometry and liquid chromatography matrix-assisted laser desorption/ionization tandem mass spectrometry. The use of two ionization/instrumental configurations led to complementary peptide identifications that increased the confidence of protein assignments. Examples that illustrate the power of this strategy are taken from two different projects: i) immunoaffinity purified complexes containing the prion protein from the murine brain, and ii) human tracheal epithelium gland secretions. In these studies, a large number of novel proteins were identified using stringent match criteria, in addition to many that had been identified in previous experiments. In the latter case, the ICAT method produced significant new information on changes that occur in protein expression levels in a patient suffering from cystic fibrosis.

Amino Acid Sequence↗

PubMed2003

Human apoC-IV: isolation, characterization, and immunochemical quantification in plasma and plasma lipoproteins.

Apolipoprotein C-IV (apoC-IV), the newest member of the low-molecular-weight apoC group, has been characterized in blood plasma of rabbits, in which it is a major proline-rich apoC component (Zhang, L-H., L. Kotite, and R. J. Havel. 1996. Identification, characterization, cloning, and expression of apoC-IV, a novel sialoglycoprotein of rabbit plasma lipoproteins. J. Biol. Chem. 271: 1776-1783). Although the decoded sequence of mouse and human apoC-IV is known, apoC-IV has not been identified in blood plasma from these or other species. Rabbit apoC-IV exists in several sialoforms, and the asialoform has an acidic isoelectric point. We show that apoC-IV is a basic protein in human, monkey, and mouse plasma, present as a minor apoC component of VLDL. Human apoC-IV, isolated from apo VLDL by DEAE-cellulose chromatography and two-dimensional electrophoresis, was identified by microsequencing four tryptic peptides. The protein exhibits two major isoforms; one is N-glycosylated, and both are variably sialylated. In normolipidemic plasma, greater than 80% of the protein is in VLDL (0.7% of total apo VLDL), with most of the remainder in HDL. The concentration of apoC-IV in the plasma and lipoproteins of rho < 1.21 g/ml is closely related to plasma triglyceride concentration up to 1,770 mg/dl, varying from 0.1-1.9 mg/dl. Neither the human nor rabbit apoC-IV gene contains a typical TATA box in the 5'-flanking region, but the 5'-untranslated region of the rabbit gene contains a unique purine-rich sequence, GGGACAG(G/A), repeated nine times in tandem, with an additional two within the 5'-flanking sequence. This sequence, functioning as a GAGA box that has been implicated in the transcription of a number of genes, may explain the higher level of expression of apoC-IV in rabbits.

5' Untranslated Regions↗

PubMed2003

Identification of novel sites of O-N-acetylglucosamine modification of serum response factor using quadrupole time-of-flight mass spectrometry.

The addition of a single N-acetylglucosamine moiety O-linked to serine and threonine residues of nuclear and cytoplasmic proteins is a widespread post-translational modification. The conventional method for detecting and locating sites of modification is through a multistep radioactivity-based approach. We have recently shown that sites of O-GlcNAc modification can be determined using quadrupole time-of-flight tandem mass spectrometry (Chalkley, R. J., and Burlingame, A. L. (2001) Identification of GlcNAcylation sites of peptides and alpha-crystallin using Q-TOF mass spectrometry. J. Am. Soc. Mass Spectrom. 12, 1106-1113). In this work utilization of this new approach has revealed previously undetected sites of O-GlcNAc modification of the transcription factor serum response factor.

Acetylglucosamine↗

PubMed2002

Factors governing the solubilization of phosphopeptides retained on ferric NTA IMAC beads and their analysis by MALDI TOFMS.

We have revisited the direct analysis experiments reported by Tomer and co-workers in the MALDI-TOFMS analysis of phosphopeptide-loaded immobilized metal ion affinity chromatography (IMAC) beads (Zhou, W.; Merrick, B. A.; Khaledi, M. G.; Tomer, K. B. J. Am. Soc. Mass Spectrom. 2000, 11, 273-282). The results described herein provide no evidence to support a laser-induced direct desorption of phosphopeptides chelated on IMAC beads. However, we have established that solubilization of mono-phosphopeptides from their immobilized Fe3+-NTA chelates does occur effectively in solutions containing certain MALDI matrices. Particularly effective is 2,5-dihydroxybenzoic acid (2,5-DHB), which apparently forms a stronger chelation complex with Fe3+-NTA than mono-phosphopeptides. With regard to the disparity observed between the low pH value of MALDI matrices (saturated 2,5-DHB(aq) approximately pH 2) and the high pH values of conventional IMAC eluents (typically above pH 7), we have also investigated the influence of eluent pH on the recovery of phosphopeptides from IMAC media. Finally, we have confirmed the importance of employing ammonium dihydrogen phosphate as buffer to achieve effective liberation of mono- and all poly-phosphopeptide species from Fe3+-NTA IMAC resin.

Amino Acid Sequence↗

PubMed2002

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.

Animals↗

PubMed2001

Functional assignment of the 20 S proteasome from Trypanosoma brucei using mass spectrometry and new bioinformatics approaches.

As experimental technologies for characterization of proteomes emerge, bioinformatic analysis of the data becomes essential. Separation and identification technologies currently based on two-dimensional gels/mass spectrometry provide the inherent analytical power required. This strategy involves protein spot digestion and accurate mass mapping together with computational interrogation of available data bases for protein functional identification. When either no exact match is found or when the possible matches only partially account for molecular weights actually observed, peptide sequencing by tandem mass spectrometry has emerged as the methodology of choice to provide the basic additional information required. To evaluate the capabilities of bioinformatics methods employed for identifying homologs of a protein of interest, we attempted to identify the major proteins from the 20 S proteasome of Trypanosoma brucei using sequence information determined using mass spectrometry. The results suggest that neither the traditional query engines, BLAST and FASTA, nor specialized software developed for analysis of sequence information obtained by mass spectrometry are able to identify even closely related sequences at statistically significant scores. To address this deficit, new bioinformatics approaches were developed for concomitant use of the multiple fragments of short sequence typically available from methods of tandem mass spectrometry. These approaches rely on the occurrence of congruence across searches of multiple fragments from a single protein. This method resulted in sharply better statistical significance values for correct hits in the data base output relative to that achieved for independent searches using single sequence fragments.

Algorithms↗

PubMed2001

Matrix-assisted laser desorption/ionization coupled with quadrupole/orthogonal acceleration time-of-flight mass spectrometry for protein discovery, identification, and structural analysis.

The design and operation of a novel UV-MALDI ionization source on a commercial QqoaTOF mass spectrometer (Applied Biosystem/MDS Sciex QSTAR Pulsar) is described. Samples are loaded on a 96-well target plate, the movement of which is under software control and can be readily automated. Unlike conventional high-energy MALDI-TOF, the ions are produced with low energies (5-10 eV) in a region of relatively low vacuum (8 mTorr). Thus, they are cooled by extensive low-energy collisions before selection in the quadrupole mass analyzer (Q1), potentially giving a quasi-continuous ion beam ideally suited to the oaTOF used for mass analysis of the fragment ions, although ion yields from individual laser shots may vary widely. Ion dissociation is induced by collisions with argon in an rf-only quadrupole cell, giving typical low-energy CID spectra for protonated peptide ions. Ions separated in the oaTOF are registered by a four-anode detector and time-to-digital converter and accumulated in "bins" that are 625 ps wide. Peak shapes depend upon the number of ion counts in adjacent bins. As expected, the accuracy of mass measurement is shown to be dependent upon the number of ions recorded for a particular peak. With internal calibration, mass accuracy better than 10 ppm is attainable for peaks that contain sufficient ions to give well-defined Gaussian profiles. By virtue of its high resolution, capability for accurate mass measurements, and sensitivity in the low-femotomole range, this instrument is ideally suited to protein identification for proteomic applications by generation of peptide tags, manual sequence interpretation, identification of modifications such as phosphorylation, and protein structural elucidation. Unlike the multiply charged ions typical of electrospray ionization, the singly charged MALDI-generated peptide ions show a linear dependence of optimal collision energy upon molecular mass, which is advantageous for automated operation. It is shown that the novel pulsing technique of this instrument that increases the sensitivity for precursor ions scans is applicable to the identification of peptides labeled with isotope-coded affinity tags.

Databases, Factual↗

PubMed2001

Identification of new JNK substrate using ATP pocket mutant JNK and a corresponding ATP analogue.

Modification of the ATP pocket on protein kinases allows selective use of an ATP analogue that exhibits high affinity for the altered kinases. Using this approach, we altered the ATP-binding site on JNK and identified N(6)-(2-phenythyl)-ATP, a modified form of ATP that exhibits high specificity and affinity for the modified, but not the wild type form, of JNK. Using modified JNK and its ATP analogue enables the detection of novel JNK substrates. Among substrates identified using this approach is heterogeneous nuclear ribonucleoprotein K, which is involved in transcription and post-transcriptional mRNA metabolism. The newly identified substrate can be phosphorylated by JNK on amino acids 216 and 353, which contribute to heterogeneous nuclear ribonucleoprotein K mediated transcriptional activities.

Adenosine Triphosphate↗