The β-lactamase gene regulator AmpR is a tetramer that recognizes and binds the D-Ala-D-Ala motif of its repressor UDP-N-acetylmuramic acid (MurNAc)-pentapeptide.

Inducible expression of chromosomal AmpC β-lactamase is a major cause of β-lactam antibiotic resistance in the Gram-negative bacteria Pseudomonas aeruginosa and Enterobacteriaceae. AmpC expression is induced by the LysR-type transcriptional regulator (LTTR) AmpR, which activates ampC expression in response to changes in peptidoglycan (PG) metabolite levels that occur during exposure to β-lactams. Under normal conditions, AmpR represses ampC transcription by binding the PG precursor UDP-N-acetylmuramic acid (MurNAc)-pentapeptide.

Tat peptide-calmodulin binding studies and bioinformatics of HIV-1 protein-calmodulin interactions.

The human immunodeficiency virus type 1 (HIV-1) genome encodes 18 proteins and 2 peptides. Four of these proteins encode high-affinity calmodulin-binding sites for which direct interactions with calmodulin have already been described. In this study, the HIV-1 proteome is queried using an algorithm that predicts calmodulin-binding sites revealing seven new putative calmodulin-binding sites including residues 34-56 of the transactivator of transcription (Tat).

Dual N- and C-terminal processing of citrus chlorophyllase precursor within the plastid membranes leads to the mature enzyme.

Chl, the central player in harvesting light energy for photosynthesis, is enzymatically degraded during natural turnover, leaf senescence, fruit ripening or following biotic/abiotic stress induction. The photodynamic properties of Chl and its metabolites call for tight regulation of the catabolic pathway enzymes to avoid accumulation of intermediate breakdown products. Chlorophyllase, the Chl dephytilation enzyme, was previously demonstrated to be an initiator of Chl breakdown when transcriptionally induced to be expressed during ethylene-induced citrus fruit color break or when heterologously expressed in different plant systems.

Predicting retention time shifts associated with variation of the gradient slope in peptide RP-HPLC.

We have developed a sequence-specific model for predicting slopes (S) in the fundamental equation of linear solvent strength theory for the reversed-phase HPLC separation of tryptic peptides detected in a typical bottom-up-proteomics experiment. These slopes control the variation in the separation selectivity observed when the physical parameters of chromatographic separation, such as gradient slope, flow rate, and column size are altered. For example, with the use of an arbitrarily chosen set of tryptic peptides with a 4-times difference in the gradient slope between two experiments, the R(2)-value of correlation between the observed retention times of identical species decreases to ~0.

Mass spectrometry following mild enzymatic digestion reveals phosphorylation of recombinant proteins in Escherichia coli through mechanisms involving direct nucleotide binding.

A straightforward method using mild enzymatic digestions combined with MALDI mass spectrometry (MS) was used to enhance determination of the multiple phosphorylation sites of a set of recombinant nucleotide-binding proteins in Escherichia coli, including kinases and cystathionine beta-synthase (CBS) domain containing proteins. The protein kinases reveal abundant phosphorylations in the kinase domains and relatively low phosphogluconoylation (258 Da) at the N-terminal His-tag. In contrast, the CBS domain-containing proteins possess a highly conserved phosphorylation in vivo at Ser-2 of the His-tag.

Urea as a protein destabilizing agent in electrospray ionisation.

Urea is well known as a denaturant of proteins, but there is also evidence that millimolar amounts of urea may in fact stabilize protein complexes. Advances in mass spectrometric analysis have given us the opportunity to test the effect of urea on several noncovalent complexes in buffered solutions. We expected to see lower charge states if folded proteins were more compact (and therefore more stable), and higher charge states if the proteins were denatured.

Practical implementation of 2D HPLC scheme with accurate peptide retention prediction in both dimensions for high-throughput bottom-up proteomics.

We describe the practical implementation of a new RP (pH 10 - pH 2) 2D HPLC-ESI/MS scheme for large-scale bottom-up analysis in proteomics. When compared to the common SCX-RP approach, it provides a higher separation efficiency in the first dimension and increases the number of identified peptides/proteins. We also employed the methodology of our sequence-specific retention calculator (SSRCalc) and developed peptide retention prediction algorithms for both LC dimensions.

Citrus chlorophyllase dynamics at ethylene-induced fruit color-break: a study of chlorophyllase expression, posttranslational processing kinetics, and in situ intracellular localization.

Fruit color-break is the visual manifestation of the developmentally regulated transition of chloroplasts to chromoplasts during fruit ripening and often involves biosynthesis of copious amounts of carotenoids concomitant with massive breakdown of chlorophyll. Regulation of chlorophyll breakdown at different physiological and developmental stages of the plant life cycle, particularly at fruit color-break, is still not well understood. Here, we present the dynamics of native chlorophyllase (Chlase) and chlorophyll breakdown in lemon (Citrus limon) fruit during ethylene-induced color-break.

Triticum mosaic virus: A New Virus Isolated from Wheat in Kansas.

In 2006, a mechanically-transmissible and previously uncharacterized virus was isolated in Kansas from wheat plants with mosaic symptoms. The physiochemical properties of the virus were examined by purification on cesium chloride density gradients, electron microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), sequencing of the nucleotides and amino acids of the coat protein, and immunological reactivity. Purified preparations contained flexuous, rod-shaped particles that resembled potyviruses.

Sequence-specific retention calculator. A family of peptide retention time prediction algorithms in reversed-phase HPLC: applicability to various chromatographic conditions and columns.

Separation selectivity of C18 reversed-phase columns from different manufacturers has been compared to evaluate the applicability of our sequence-specific retention calculator (SSRCalc) peptide retention prediction algorithms. Three different versions of SSRCalc are currently in use: 300-A pore size sorbents (TFA as ion-pairing modifier, pH 2), 100 A (TFA, pH 2), and 100 A (pH 10), which have been applied for the separation of randomly chosen mixture of tryptic peptides. The major factor affecting separation selectivity of C18 sorbents was found to be apparent pore size, while differences in end-capping chemistry do not introduce a significant impact.

Formation of (bn-1 + H2O) ions by collisional activation of MALDI-formed peptide [M + H]+ ions in a QqTOF mass spectrometer.

Collisional activation of [M + H](+) parent ions from peptides of n amino acid residues may yield a rearrangement that involves loss of the C-terminal amino acid residue to produce (b(n-1) + H(2)O) daughters. We have studied this reaction by a retrospective examination of the m/z spectra of two collections of data. The first set comprised 398 peptides from coat protein digests of a number of plant viruses by various enzymes, where conditions in the tryptic digests were chosen so as to produce many missed cleavages.

Deamidation of -Asn-Gly- sequences during sample preparation for proteomics: Consequences for MALDI and HPLC-MALDI analysis.

We find that peptides containing -Asn-Gly- sequences typically show approximately 70-80% degree of deamidation after standard overnight (approximately 12 h) tryptic digestion at 37 degrees C. This emphasizes the need for more detailed information about the deamidation reaction in -Asn-Gly- sequences, in which two deamidated species are produced, one containing an aspartic acid (-Asp-Gly-) residue and the other containing an isoaspartic acid (-betaAsp-Gly-) residue. For the peptide SLNGEWR (54-60 beta-galactosidase, E.

Use of peptide retention time prediction for protein identification by off-line reversed-phase HPLC-MALDI MS/MS.

A new algorithm, sequence-specific retention calculator, was developed to predict retention time of tryptic peptides during RP HPLC fractionation on C18, 300-A pore size columns. Correlations of up to approximately 0.98 R2 value were obtained for a test library of approximately 2000 peptides and approximately 0.

Method for estimating the isotopic distributions of metabolically labeled proteins by MALDI-TOFMS: application to NMR samples.

We have developed an efficient method of estimating metabolic incorporation of heavy isotopes into proteins, including those where a single amino acid carries the label. The protein is digested with trypsin, and the resulting peptide mixture is examined directly by MALDI-TOF mass spectrometry. Peptides are chosen for analysis if they contain one or more labeled atoms and also exhibit clearly separated mass spectra.

MALDI QqTOF MS combined with off-line HPLC for characterization of protein primary structure and post-translational modifications.

The addition of off-line high-performance liquid chromatography to matrix-assisted laser desorption/ionization mass spectrometry greatly reduces congestion in the mass spectra, and also provides complete decoupling of the separation process from mass detection and measurement. This removes the time constraints inherent in on-line coupling, and so enables the detailed mass-spectrometric study of samples at later times. We describe here our use of this method to successfully characterize two "unknown" protein mixtures that were set as problems by the ABRF Proteomics Research Group (PRG) in the years 2003 and 2004.

Determination and characterization of site-specific N-glycosylation using MALDI-Qq-TOF tandem mass spectrometry: case study with a plant protease.

MALDI tandem mass spectrometry analysis on a hybrid quadrupole-quadrupole time-of-flight (Qq-TOF) instrument was used in combination with two-dimensional gel electrophoresis, proteolytic digestion, and liquid chromatography for identification and structural characterization of glycosylation in a novel glycoprotein, pathogenesis-related subtilisin-like proteinase P69B from tomato. Glycopeptide fractions from microcolumn reversed-phase HPLC deposited on MALDI targets were identified from MS by their specific m/z spacing patterns (203, 162, 146 u) between glycoforms. In most cases, MS/MS spectra of [M + H]+ ions of glycopeptides featured peaks useful for determining sugar compositions, peptide sequences, and thus probable glycosylation sites.

Hybrid quadrupole/time-of-flight mass spectrometers for analysis of biomolecules.

The basic principles of quadrupole/time-of-flight (TOF) mass spectrometers are discussed. These instruments can be used for ions produced either by electrospray ionization (ESI) or by matrix-assisted laser desorption ionization (MALDI). In the most common configuration, the functions of collisional cooling, parent ion selection, and collision-induced dissociation are carried out successively in three separate quadrupoles.

Association of a Virus with Wheat Displaying Yellow Head Disease Symptoms in the Great Plains.

Wheat with yellow head disease (YHD) (yellow heads and mosaic leaf symptoms) has been observed in Kansas since 1997. A pathogen was transmitted from the infected wheat to maize by vascular puncture inoculation and to Nicotiana benthamiana by rub inoculation. The original infected wheat and infected maize and N.

Multiple equilibria of the Escherichia coli chaperonin GroES revealed by mass spectrometry.

Nanospray time-of-flight mass spectrometry has been used to study the assembly of the heptamer of the Escherichia coli cochaperonin protein GroES, a system previously described as a monomer-heptamer equilibrium. In addition to the monomers and heptamers, we have found measurable amounts of dimers and hexamers, the presence of which suggests the following mechanism for heptamer assembly: 2 Monomers <--> Dimer; 3 Dimers <--> Hexamer; Hexamer + Monomer <--> Heptamer. Equilibrium constants for each of these steps, and an overall constant for the Monomer <--> Heptamer equilibrium, have been estimated from the data.

Global and site-specific detection of human integrin alpha 5 beta 1 glycosylation using tandem mass spectrometry and the StrOligo algorithm.

Glycans are oligosaccharides associated with proteins, and are known to confer specific functions and conformations on glycoproteins. As protein tridimensional structures are related to function, the study of glycans and their impact on protein folding can provide important information to the field of proteomics. The subdiscipline of glycomics (or glycoproteomics) is rapidly growing in importance as glycans in proteins have shown to be involved in protein-protein or protein-(drug, virus, antibody) interactions.

Biochemical and proteomics approaches to characterize topoisomerase IIalpha cysteines and DNA as targets responsible for cisplatin-induced inhibition of topoisomerase IIalpha.

Cisplatin was shown to strongly inhibit the decatenation and relaxation activity of isolated human DNA topoisomerase IIalpha. This inhibition was not accompanied by stabilization of a covalent topoisomerase IIalpha-DNA intermediate. Pretreatment of kinetoplast plasmid DNA (kDNA) or pBR322 DNA with submicromolar concentrations of cisplatin quickly rendered these substrates incompetent in the topoisomerase IIalpha catalytic assay.

Site-specific N-glycosylation analysis: matrix-assisted laser desorption/ionization quadrupole-quadrupole time-of-flight tandem mass spectral signatures for recognition and identification of glycopeptides.

The identification of glycosylation sites in proteins is often possible through a combination of proteolytic digestion, separation, mass spectrometry (MS) and tandem MS (MS/MS). Liquid chromatography (LC) in combination with MS/MS has been a reliable method for detecting glycopeptides in digestion mixtures, and for assigning glycosylation sites and glycopeptide sequences. Direct interfacing of LC with MS relies on electrospray ionization, which produces ions with two, three or four charges for most proteolytic peptides and glycopeptides.

Lectin affinity as an approach to the proteomic analysis of membrane glycoproteins.

The aim was to determine the proportion of membrane glycoproteins captured using concanavalin A or wheat germ agglutinin lectin affinity chromatography. Digests of the isolated proteins were separated by reversed-phase liquid chromatography and analyzed by matrix-assisted laser desorption tandem mass spectrometry. The two lectins identified different groups of proteins with a broad range of molecular mass and p/ values, including a number of proteins that overlapped the two groups.

Device for the reversed-phase separation and on-target deposition of peptides incorporating a hydrophobic sample barrier for matrix-assisted laser desorption/ionization mass spectrometry.

The separation of peptide mixtures from proteolytic cleavage is often necessary prior to mass spectrometry (MS) to enhance sensitivity and peptide mapping coverage. When buffers, salts, and other higher abundance peptides/contaminants are present, competition for charge during the electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) processes can lead to ion suppression for the targeted analyte(s). In this note, a simple reversed-phase microcolumn sample separation and deposition device (Sep-Dep) is described.

Application of the StrOligo algorithm for the automated structure assignment of complex N-linked glycans from glycoproteins using tandem mass spectrometry.

Oligosaccharides associated with proteins are known to give these molecules specific conformations and functions. Analysis of proteins would not be complete without studying the glycans. However, high-throughput techniques in proteomics will soon overwhelm the current capacity of methods if no automation is incorporated into glycomics.