Identification of Variants of the High Plains virus Infecting Wheat in Kansas.

The properties of two virus isolates (U04-82 and U04-83) obtained from two wheat (Triticum aestivum) plants expressing mosaic symptoms were investigated using enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), time-of-flight mass spectrometry (TOFMS), and infection of wheat with resistance to Wheat streak mosaic virus (WSMV). The coat protein mass was estimated by SDS-PAGE as approximately 32 kDa for U04-82 and 30 kDa for U04-83. The amino acid sequence of the coat protein of U04-82 was 99.6 and 85.5% identical to two isolates, ABC58222 and TX96, respectively, of High Plains virus (HPV) described from Texas. U04-82 was transmitted by wheat curl mites and caused significant yield reductions in wheat resistant to WSMV. U04-83 was actually two distinct virus isolates whose capsid protein amino acid sequences were only 57 and 50% similar to that of TX96. Antiserum prepared to a synthetic peptide from the sequence of the U04-83 isolate recognized the two U04-83 isolates, but not the U04-82 isolate.

Characterization of a distinct Johnsongrass mosaic virus strain isolated from sorghum in Nigeria.

A virus isolated from sorghum in Nigeria has been partially characterized. It was tested by enzyme-linked immunosorbent assay (ELISA) using antisera to Maize dwarf mosaic virus, Johnsongrass mosaic virus (JGMV), Sugarcane mosaic virus strain-MDB, Sorghum mosaic virus, and Zea mosaic virus. A partial host range, symptom phenotypes for selected sorghum lines, and the mass of the coat protein (CP) subunit was analyzed by sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and its amino acid (aa) sequence determined by time-of-flight mass spectrometry (TOFMS). The Nigerian isolate was positive in ELISA to only JGMV antiserum. It infected sorghum and smooth brome but not oat or johnsongrass. It caused necrosis in 12 of 13 tested sorghum lines, while the USA JGMV isolate caused necrosis in only one sorghum line. In SDS-PAGE, the mass of the Nigerian virus CP was 3,000 Da smaller than that of JGMV-MDO. Moreover, TOFMS analyses showed that, while residues 1-7 of the CP aa sequence were identical to those of JGMV (GenBank #A27631), and residues 57-293 were almost identical to residues 67-303 of JGMV, the intermediate region exhibited significant differences, including a 10 aa deletion. These data indicate that the virus should be considered a distinct isolate of JGMV (JGMV-N) and expands the known range of JGMV to Africa.

An improved model for prediction of retention times of tryptic peptides in ion pair reversed-phase HPLC: its application to protein peptide mapping by off-line HPLC-MALDI MS.

The proposed model is based on the measurement of the retention times of 346 tryptic peptides in the 560- to 4,000-Da mass range, derived from a mixture of 17 protein digests. These peptides were measured in HPLC-MALDI MS runs, with peptide identities confirmed by MS/MS. The model relies on summation of the retention coefficients of the individual amino acids, as in previous approaches, but additional terms are introduced that depend on the retention coefficients for amino acids at the N-terminal of the peptide. In the 17-protein mixture, optimization of two sets of coefficients, along with additional compensation for peptide length and hydrophobicity, yielded a linear dependence of retention time on hydrophobicity, with an R2 value about 0.94. The predictive capability of the model was used to distinguish peptides with close m/z values and for detailed peptide mapping of selected proteins. Its applicability was tested on columns of different sizes, from nano- to narrow-bore, and for direct sample injection, or injection via a pre-column. It can be used for accurate prediction of retention times for tryptic peptides on reversed-phase (300-A pore size) columns of different sizes with a linear water-ACN gradient and with TFA as the ion-pairing modifier.

Biological and Molecular Variability Among High Plains virus Isolates.

The High Plains virus (HPV), vectored by the wheat curl mite (WCM) (Aceria tosichella), causes a severe disease of maize (Zea mays) in the U. S. High Plains. In the present study, five HPV isolates from five states were separated from co-infecting Wheat streak mosaic virus and their molecular and biological variability studied. Molecular studies involved time-of-flight mass spectrometry (TOFMS) to determine amino acid sequence variability of the 32-kDa nucleoprotein (32 np) of the isolates. Biological studies involved testing the ability of the five HPV isolates to infect a maize line previously shown to have resistance. Inoculations of the HPV isolates were conducted using vascular puncture inoculation (VPI) and viruliferous WCM. TOFMS analyses demonstrated an 18-amino acid sequence in the isolates at the N-terminus of the 32 np, the presence of amino acid sequence differences among the isolates, and variability among amino acid sequences of the 32 np of some isolates. Three of the five HPV isolates infected the resistant maize inbred, B73, using VPI, and two of the same three HPV isolates infected this line using WCM inoculation, albeit low numbers of plants were infected by each technique.

Hepatocyte [3H]-palmitate uptake: effect of albumin surface charge modification.

The role of plasma proteins on the cellular uptake of lipophilic substrates has perplexed investigators for many years. We tested the hypothesis that an ionic interaction between the protein-ligand complex and hepatocyte surface may be responsible for supplying more ligand to the cell for uptake. The surface-charged groups on albumin were modified to yield proteins having a range of isoelectric points (ALB, ALBs, ALBm, ALBe had values of 4.8-5.0, 4.5-4.7, 3.0-3.5, 8.4-8.6, respectively). [3H]-Palmitate uptake studies were performed with adult rat hepatocyte suspensions using similar unbound ligand fractions in the presence of the different binding proteins. Mass spectrometry, isoelectric focusing (pI), and heptane:water partitioning were used to determine protein molecular weight, pI, and protein-palmitate equilibrium binding constant, respectively. Hepatocyte [3H]-palmitate clearance in the presence of ALBs and ALBm were significantly lower (p < 0.05) than ALB, whereas [3H]-palmitate clearance in the presence of ALBe was significantly higher (p < 0.05) than ALB. The data were consistent with the notion that ionic interactions between extracellular protein-ligand complexes and the hepatocyte surface facilitate the uptake of long-chain fatty acids.

Matrix-assisted laser desorption/ionization quadrupole time-of-flight mass spectrometry: an elegant tool for peptidomics.

A Matrix-assisted laser desorption/ionization hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer was employed to acquire neuropeptide mass spectra, directly from neuropeptide secreting tissue deposited on the sample target, in the presence of dihydroxybenzoic acid as matrix. The cockroach corpus cardiacum served as model neuroendocrine tissue. Twelve neuropeptide ion peaks, with mass-to-charge ratio values ranging between 800 and 3,000 Da were selected for tandem mass spectrometry. All peptides below 1,600 Da could be fully sequenced; tandem mass spectrometry analysis of the remaining (three) largest peptides resulted in (limited) sequence tags, which, also due to unavailability of an appropriate neuropeptide structure database, did not allow complete structure elucidation.

Mass spectrometric study of the Escherichia coli repressor proteins, Ic1R and Gc1R, and their complexes with DNA.

In Escherichia coli, the IclR protein regulates both the aceBAK operon and its own synthesis. Database homology searches have identified many IclR-like proteins, now known as the IclR family, which can be identified by a conserved C-terminal region. We have cloned and purified one of these proteins, which we have named GclR (glyoxylate carboligase repressor). Although purification is straightforward, both the IclR and GclR proteins are difficult to manipulate, requiring high salt (up to 0.6 M KCl) for solubility. With the advent of nanospray ionization, we could transfer the proteins into much higher concentrations of volatile buffer than had been practical with ordinary electrospray. In 0.5 M ammonium bicarbonate buffer, both proteins were stable as tetramers, with a small amount of dimer. In a separate experiment, we found that IclR protein selected from a random pool a sequence which matched exactly that of the presumed binding region of the GclR protein, although IclR does not regulate the gcl gene. We designed a 29 bp synthetic DNA to which IclR and GclR bind, and with which we were able to form noncovalent DNA-protein complexes for further mass spectrometry analysis. These complexes were far more stable than the proteins alone, and we have evidence of a stoichiometry which has not been described previously with (protein monomer : dsDNA) = (4 : 1).

Archived polyacrylamide gels as a resource for proteome characterization by mass spectrometry.

Mass spectrometry was applied to identify protein spots excised from an archived two-dimensional polyacrylamide gel that had been dried and stored for eight years at room temperature. All proteins were successfully identified. Detailed characterization of protein digests by matrix-assisted laser desorption/ionization (MALDI) peptide mapping, nanoelectrospray tandem mass spectrometry and MALDI-quadrupole time-of-flight mass spectrometry revealed no evidence of protein degradation or modifications that could hamper identification of proteins in a sequence database. The experiment with a model protein demonstrated that the pattern of tryptic peptides and the yield of individual peptides were not noticeably changed in the in-gel digest of the archived protein spot compared to the digest of the spot excised from a fresh gel. Thus, the characterization of "archived proteomes" has the potential to advance proteomic research without repeating "wet" biochemistry experiments, that had been perfected in the laboratory years ago.

Charting the proteomes of organisms with unsequenced genomes by MALDI-quadrupole time-of-flight mass spectrometry and BLAST homology searching.

MALDI-quadrupole time-of-flight mass spectrometry was applied to identify proteins from organisms whose genomes are still unknown. The identification was carried out by successively searching a sequence database-first with a peptide mass fingerprint, then with a packet of noninterpreted MS/MS spectra, and finally with peptide sequences obtained by automated interpretation of the MS/MS spectra. A "MS BLAST" homology searching protocol was developed to overcome specific limitations imposed by mass spectrometric data, such as the limited accuracy of de novo sequence predictions. This approach was tested in a small-scale proteomic project involving the identification of 15 bands of gel-separated proteins from the methylotrophic yeast Pichia pastoris, whose genome has not yet been sequenced and which is only distantly related to other fungi.

Quantitative proteomic analysis using a MALDI quadrupole time-of-flight mass spectrometer.

We describe an approach to the quantitative analysis of complex protein mixtures using a MALDI quadrupole time-of-flight (MALDI QqTOF) mass spectrometer and isotope coded affinity tag reagents (Gygi, S. P.; et al. Nat. Biotechnol. 1999, 17, 994-9.). Proteins in mixtures are first labeled on cysteinyl residues using an isotope coded affinity tag reagent, the proteins are enzymatically digested, and the labeled peptides are purified using a multidimensional separation procedure, with the last step being the elution of the labeled peptides from a microcapillary reversed-phase liquid chromatography column directly onto a MALDI sample target. After addition of matrix, the sample spots are analyzed using a MALDI QqTOF mass spectrometer, by first obtaining a mass spectrum of the peptides in each sample spot in order to quantify the ratio of abundance of pairs of isotopically tagged peptides, followed by tandem mass spectrometric analysis to ascertain the sequence of selected peptides for protein identification. The effectiveness of this approach is demonstrated in the quantification and identification of peptides from a control mixture of proteins of known relative concentrations and also in the comparative analysis of protein expression in Saccharomyces cerevisiae grown on two different carbon sources.

Determination of the complete amino acid sequence for the coat protein of brome mosaic virus by time-of-flight mass spectrometry. Evidence for mutations associated with change of propagation host.

Time-of-flight mass spectrometry (TOFMS) has been applied to determine the complete coat protein amino acid sequences of a number of distinct brome mosaic virus (BMV) isolates. Ionization was carried out by both electrospray ionization and matrix-assisted laser desorption/ionization (MALDI). After determining overall coat protein masses, the proteins were digested with trypsin or Lys-C proteinases, and the digestion products were analyzed in a MALDI QqTOF mass spectrometer. The N terminus of the coat protein was found to be acetylated in each BMV isolate analyzed. In one isolate (BMV-Valverde), the amino acid sequence was identical to that predicted from the cDNA sequence of the "type" isolate, but deviations from the predicted amino acid sequence were observed for all the other isolates analyzed. When isolates were propagated in different host taxa, modified coat protein sequences were observed in some cases, along with the original sequence. Sequencing by TOFMS may therefore provide a basis for monitoring the effects of host passaging on a virus at the molecular level. Such TOFMS-based analyses assess the complete profiles of coat protein sequences actually present in infected tissues. They are therefore not subject to the selection biases inherent in deducing such sequences from reverse-transcribed viral RNA and cloning the resulting cDNA.

On-membrane digestion of beta-casein for determination of phosphorylation sites by matrix-assisted laser desorption/ionization quadrupole/time-of-flight mass spectrometry.

This article discusses the features of a newly developed matrix-assisted laser desorption/ionization quadrupole/time-of-flight (MALDI-QqTOF) mass spectrometer that is useful in the analysis of phosphorylated peptides. Aliquots of beta-casein, a commonly used phosphorylated protein standard, were digested with trypsin directly on a non-porous polyurethane membrane used as sample support in MALDI-QqTOF mass spectrometry (MS) experiments. Although a complete peptide map was obtained, it was difficult to obtain sequence information for some of the tryptic fragments, in particular T1-2, which bears four phosphate groups and is thus difficult to ionize in positive mode. This article focuses on the sequencing of this particular fragment by comparing MS/MS spectra obtained using different precursor ions. These precursors associated with T1-2 were [M + H](+), [M + H](2+), and [M + H - nH(3)PO(4)](+) ions. Typically, phosphorylated ions showed facile unimolecular losses of phosphoric acid moieties, and produced limited backbone fragmentation. The abundance of [M + H](2+) ions of T1-2 in the full mass spectrum was low relative to that of [M + H](+). [M + H - 4H(3)PO(4)](+) ions as MS/MS precursors underwent backbone fragmentations, with phosphoserine residues transformed into dehydroalanines or serines. Unusual b + 18 u fragments were observed, although only for segments with previously phosphorylated serines. These partly interfered with c-ions, and were noticeable due to overlapping isotopic envelopes. It was possible to establish the sequence of phosphorylated tryptic fragment T1-2 and the location of phosphate groups using the mass of dehydroalanine residues (69 Da) and b + 18 u fragments as markers. All MS and MS/MS spectra obtained with fully phosphorylated beta-casein were compared with spectra acquired with dephosphorylated beta-casein obtained commercially. These comparisons helped assess the spectral differences caused by the presence of phosphate groups. Also, they highlighted the potential usefulness of conducting dephosphorylation directly on the probe prior to MALDI analysis in future studies.

Total chemical synthesis and chemotactic activity of human S100A12 (EN-RAGE).

Human S100A12 (extracellular newly identified RAGE (receptor for advanced glycosylation end products)-binding protein), a new member of the S100 family of EF-hand calcium-binding proteins, was chemically synthesised using highly optimised 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/tert-butoxycarbonyl in situ neutralisation solid-phase chemistry. Circular dichroism studies indicated that CaCl(2) decreased the helical content by 27% whereas helicity was marginally increased by ZnCl(2). The propensity of S100A12 to dimerise was examined by electrospray ionisation time-of-flight mass spectrometry which clearly demonstrated the prevalence of the non-covalent homodimer (20890 Da). Importantly, synthetic human S100A12 in the nanomolar range was chemotactic for neutrophils and macrophages in vitro.

Characterization of plasma proteins adsorbed onto biomaterials. By MALDI-TOFMS.

The analysis of plasma proteins adsorbed onto a polyurethane (PU) biomaterial was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). This article marks the first study on MALDI-TOFMS analysis of multiple proteins adsorbed from plasma, in vitro, onto the surface of a biomaterial to easily enable their characterization. Plasma standards from three different hosts were placed in contact with non-porous PU, a model biomaterial. Following the use of washing protocols developed in our laboratory, the biomaterial was analyzed, directly, with MALDI-TOFMS. Proteins with molecular weights (Mr) ranging from ca. 6.5 to 150 kDa were observed in the mass spectra and characterized upon comparison with proteins of known Mr. The proteins observed were tentatively identified as those known to adsorb onto PU, both in vitro and in vivo. In an attempt to model in vivo sorption, the PU biomaterial was exposed to freshly collected canine plasma, in vitro, for different lengths of time. Corresponding MALDI-TOFMS spectra displayed increasing protein signal for a number of different proteins with increasing times of exposure to plasma. This method provided qualitative and semi-quantitative analysis of the proteins adsorbed onto the biomaterial surface.

MALDI quadrupole time-of-flight mass spectrometry: a powerful tool for proteomic research.

A MALDI QqTOF mass spectrometer has been used to identify proteins separated by one-dimensional or two-dimensional gel electrophoresis at the femtomole level. The high mass resolution and the high mass accuracy of this instrument in both MS and MS/MS modes allow identification of a protein either by peptide mass fingerprinting of the protein digest or from tandem mass spectra acquired by collision-induced dissociation of individual peptide precursors. A peptide mass map of the digest and tandem mass spectra of multiple peptide precursor ions can be acquired from the same sample in the course of a single experiment. Database searching and acquisition of MS and MS/MS spectra can be combined in an interactive fashion, increasing the information value of the analytical data. The approach has demonstrated its usefulness in the comprehensive characterization of protein in-gel digests, in the dissection of complex protein mixtures, and in sequencing of a low molecular weight integral membrane protein. Proteins can be identified in all types of sequence databases, including an EST database. Thus, MALDI QqTOF mass spectrometry promises to have remarkable potential for advancing proteomic research.

Characterization of a novel potyvirus isolated from maize in Israel.

A potyvirus (proposed name of Zea mosaic virus [ZeMV]) isolated from maize in Israel was analyzed by serology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of capsid proteins, symptomatology, and sequencing. Parts of the nuclear inclusion b, coat protein, and 3' regions were sequenced; the amino acid sequence of ZeMV capsid was determined by time-of-flight mass spectrometry (TOFMS). The results of these analyses were compared with those of similar analyses of the following potyviruses: Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus strain MDB (SCMV-MDB), Johnsongrass mosaic virus(JGMV), Sorghum mosaic virus (SrMV), and an isolate of MDMV from Israel. Indirect enzyme-linked immunosorbent assay using ZeMV antiserum detected only ZeMV, and reciprocal tests using MDMV, JGMV, or SrMV antisera failed to detect ZeMV. ZeMV cross-reacted weakly when SCMV-MDB antiserum was used. The mass of ZeMV capsid was determined to be 36,810 Da by SDS-PAGE and 34,216 Da by TOFMS. The ZeMV systemically infected johnsongrass (Sorghum halepense), but did not infect oat (Avena sativa), pearl millet (Pennisetum glaucum), barley (Hordeum vulgare), or rye (Secale cereale). Necrosis was caused in 19 sorghum lines by SrMV, in 15 by ZeMV, in 14 by MDMV, and in 5 by JGMV and SCMV-MDB. The nucleic acid and amino acid sequences of ZeMV clearly showed that it is not a strain of JGMV, MDMV, SCMV, or SrMV.

Structural and functional characterizations of the proteasome-activating protein PA26 from Trypanosoma brucei.

The activated 20 S proteasome, which has been found only in mammalian cells, is composed of two heptamer rings of an activator protein on each end of the 20 S proteasome and is inducible by interferon-gamma. A 20 S proteasome has been recently identified in a protozoan pathogen Trypanosoma brucei, but there has been no experimental evidence yet for the presence of a 26 S proteasome. Instead, an activated form of 20 S proteasome was isolated from this organism, which has significantly enhanced peptidase activities. It consists of an additional activator protein with an estimated molecular mass of 26 kDa (PA26) (To, W. Y., and Wang, C. C. (1997) FEBS Lett. 404, 253-262). The profile and sequences of tryptic peptides from PA26 were determined by mass spectrometry; no matches were found in the data base. The peptide sequences were used in reverse transcriptase-polymerase chain reaction to isolate a full-length cDNA clone encoding PA26. The protein sequence thus derived from it indicates little sequence identity with those of mammalian activator proteins PA28 alpha, beta, or gamma. There is only a single copy of PA26 gene in T. brucei. Purified recombinant PA26 polymerizes spontaneously to form heptamer ring with an outer diameter of 8.5 nm. The ring binds and activates 20 S proteasomes from T. brucei as well as rat, whereas human PA28alpha can neither bind nor activate T. brucei 20 S proteasome. The former is thus apparently more ubiquitous than PA28 in its capability of binding to and activating 20 S proteasomes. Its presence in T. brucei may also suggest a more ancient origin of proteasome activator proteins and a much wider involvement in protein degradation among other eukaryotic organisms than was originally envisaged.

Cerebroside sulfate activator protein (Saposin B): chromatographic and electrospray mass spectrometric properties.

Cerebroside sulfate activator protein is a small, heat-stable protein that is exceptionally resistant to proteolytic attack. This protein is essential for the catabolism of cerebroside sulfate and several other glycosphingolipids. Protein purified from pig kidney and human urine was extensively characterized by reversed-phase liquid chromatography and electrospray mass spectrometry. These two sources revealed 20 and 18 different molecular isoforms of the protein, respectively. Plausible explanations of the structures of the majority of these isoforms can be made on the basis of accurate molecular mass assignments. The reversed-phase chromatographic and electrospray mass spectrometric properties of enzymatically deglycosylated and disulfide-reduced protein were also compared. In addition to a demonstration of the power of electrospray ionization mass spectrometry for revealing a wealth of information on protein microheterogeneity and structural detail, the results also demonstrate the utility of this technique for monitoring spontaneous chemical and enzymatically mediated changes that occur as a result of metabolic processing and protein purification.

Proteasome activator 11S REG or PA28: recombinant REG alpha/REG beta hetero-oligomers are heptamers.

The proteasome activator 11S REG or PA28 is a conical molecule composed of two homologous subunits, REG alpha and REG beta. Recombinant REG alpha forms a heptamer, whereas recombinant REG beta is a monomer. When mixed with REG beta, a monomeric REG alpha mutant (N50Y) forms an active hetero-oligomer in which the molar ratio of REG beta to REG alpha(N50Y) is close to 1.3. This apparent stoichiometry is consistent with the REG alpha(N50Y)/REG beta hetero-oligomer being a heptamer composed of three alpha and four beta subunits. Chemical cross-linking of the alpha/beta oligomers revealed the presence of REG alpha-REG beta and REG beta-REG beta dimers, but REG alpha-REG alpha dimers were not detected. The mass of the REG alpha(N50Y)/REG beta hetero-oligomer determined by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) is 194 871 +/- 40 Da in good agreement with the theoretical mass of 194 856 Da for an alpha 3 beta 4 heptamer. Hexamers were not observed in the mass spectrum. For wild-type REG subunits coexpressed in bacteria cells at an apparent beta/alpha molar ratio of approximately 1.2, the resulting hetero-oligomers observed by ESI-TOF MS were again predominantly alpha 3 beta 4 heptamers, with trace amounts of alpha 4 beta heptamers also present. On the other hand, the mass spectrum contained a mixture of alpha 7, alpha 6 beta 1, alpha 5 beta 2, and alpha 4 beta 3 heptamers when the REG beta/REG alpha ratio was 0.1. Thus, formation of heptamers is an intrinsic property of recombinant REG alpha and REG beta subunits. On the basis of these results, we propose that 11S REG purified directly from eukaryotic cells is also heptameric, likely alpha 3 beta 4 or a mixture of alpha 3 beta 4 and alpha 4 beta 3 species.