James D. Pearson

Single ribosomal protein mutations in antibiotic-resistant bacteria analyzed by mass spectrometry.

ABSTRACT Mutations in several ribosomal proteins are known to be related to antibiotic resistance. For several strains of Escherichia coli, the mutated protein is known but the amino acid actually altered has not been documented. Characterization of these determinants for antibiotic resistance in proteins will further the understanding of the precise mechanism of the antibiotic action as well as provide markers for resistance. Mass spectrometry can be used as a valuable tool to rapidly locate and characterize mutant proteins by using a small amount of material. We have used electrospray and matrix-assisted laser desorption ionization–time of flight (MALDI–TOF) mass spectrometry to map out all 56 ribosomal proteins in E. coli based on intact molecular masses. We used this fingerprinting approach to locate variants of ribosomal proteins displaying a change in mass. In particular we have studied proteins responsible for streptomycin, erythromycin, and spectinomycin resistance in three strains of E. coli, and then we characterized each mutation responsible for resistance by analyzing tryptic peptides of these proteins by using MALDI-TOF and nanoelectrospray tandem mass spectrometry. The results provided markers for antibiotic resistance and demonstrated that mass spectrometry can be used to rapidly investigate changes in individual proteins from a complex with picomole amounts of protein.

High-performance liquid chromatography column length designed for submicrogram scale protein isolation.

High-performance liquid chromatography of small amounts of protein was readdressed with respect to current gas-phase sequencing technology. Useful primary sequence information can be obtained from as little as 5-100 pmol of material. This corresponds on a mass level to the nanogram to microgram range where, unfortunately, HPLC columns often give low recoveries depending on the size and surface hydrophobicity of the peptide or protein. It was rationalized in this study that reduced column length could have a beneficial effect on recovery without significant loss of resolution. To demonstrate this, six HPLC columns ranging from 0.2 to 25 cm in length were made and evaluated in terms of protein loading and resolution. Column lengths of less than 1 cm were found to increase recovery of surface hydrophobic proteins without loss of resolution, as shown for a standard protein profile. These columns resolve proteins best when loaded with less than 10 micrograms, with recoveries greater than 90%. All column internal diameters were at least 4.1 mm so that standard HPLC pumps could be used to generate gradients.

A nonurea electrophoretic gel system for resolution of polypeptides of Mr 2000 to Mr 200,000

A sodium dodecyl sulfate-polyacrylamide gel electrophoresis system which resolves proteins and peptides from Mr 2000 to Mr 200,000 is described. Gradients of polyacrylamide, crosslinker, and glycerol buffered in Tris-phosphate (pH 6.8) are employed. Neither urea nor a stacking gel is required. This system has been used to separate molecules below Mr 3000 which differed by only seven amino acid residues, yet has the capacity to survey masses up to Mr 200,000 on the same gel. Examples are given for separations of myoglobin cyanogen bromide fragments and adrenocorticotropin peptides. Utilizing the same gradient slab gel system in tandem with isoelectric focusing, a two-dimensional separation pattern of mammalian liver cell lysate is shown. A comparison of two different silver stain methods with this system is also given.

CHARACTERIZATION OF A NOVEL ZINC BINDING SITE OF PROTEIN KINASE C INHIBITOR-1

The zinc‐binding properties of an endogenous protein inhibitor of protein kinase C was studied. Equilibrium gel penetration revealed that 1 mol of this protein binds 0.97 mol of zinc with a dissociation constant of 4.3 μM. The site of zinc‐binding. MVVNEGSDGGQSVYHVHLHVLGGR, was indentified by a multi‐step process consisting of tryptic digestion, fragment isolation, transfer to nitrocellulose, and hybridization with 65ZnCl2. Binding of 65ZnCl2 to selected synthetic fragments further localized the site of interaction to the sequence QSVYHVHLHVL. This region contains 3 closely positioned histidine residues and represent a novel zinc‐binding site.

The in vitro phosphorylation of calmodulin by the insulin receptor tyrosine kinase.

Calmodulin, a ubiquitous Ca2+-binding regulatory protein, is phosphorylated exclusively on tyrosine-99 in an insulin-dependent manner by wheat germ lectin-purified preparations of insulin receptors from rat adipocyte plasma membranes. Calmodulin is phosphorylated in the presence of polylysine, histone Hf2b, and protamine sulfate, but not in the absence of these cofactors or in the presence of other basic compounds known to interact with calmodulin, such as mellitin, myelin basic protein, chlorpromazine, trifluoperazine, substance P, glucagon, polyarginine, mastoparin, beta-endorphin, spermine, spermidine, and putrescine. The incorporation of 32P into calmodulin, expressed in terms of moles of phosphate per moles of calmodulin and assayed at calmodulin concentrations of 1.2 and 0.06 microM, is 0.023 + 0.002 and 0.046 + 0.006, respectively. This low stoichiometry is likely due to the relative impurity of the receptor preparation, as similar studies not shown here, using highly purified human insulin receptors, yield a stoichiometry of 1 mol phosphate/mol calmodulin. The time course of phosphorylation is characterized by a short initial lag phase of approximately 5 min, a rapid linear rate from approximately 5 to 40 min, with a steady state of 32P incorporation being approached at approximately 60 min. The K0.5 for ATP is 104 + 18 microM. Phosphorylated calmodulin is partially purified by HPLC on a C4 column using a trifluoroacetic acid/acetonitrile gradient solvent system. Phosphoamino acid analysis and limited thrombin digestion were used to determine that the site of insulin-induced phosphorylation of calmodulin is exclusively on tyrosine-99 regardless of the basic protein cofactor used. Phosphorylated calmodulin does not exhibit the characteristic Ca2+ shift normally observed with calmodulin in electrophoretic gels, an observation that is consistent with this modification affecting the biological activity of the molecule. Thus, the tyrosine phosphorylation of calmodulin represents a potentially important post-translational modification altering calmodulins ability to regulate a variety of enzymes involved in growth, differentiation, and metabolic regulation.

Separation and purification of S49 mouse lymphoma histones by reversed-phase high-performance liquid chromatography

A rapid reversed-phase high-performance liquid chromatography procedure for the fractionation of histones from S49 mouse lymphoma cells is reported. The system utilizes a Vydac C4 macroporous column, heptafluorobutyric acid as solubilizing and ion-pairing agent, and an acetonitrile gradient. All five histone classes and several subclass species are separated, including two H1 species, H2B, two H2A species, H4, and two H3 species. Analytical to multimilligram semipreparative scale fractionations are demonstrated while maintaining resolution of all histone types.

Insulin-like effects of histones H3 and H4 on isolated rat adipocytes

Crude preparations of histones had insulin-like actions in isolated adipocytes. This activity was attributed to the arginine-rich histones, H3 and H4. The metabolic effects of purified H3 and H4 on isolated adipocytes were similar to those of insulin in a number of respects. Like insulin, H3 and H4 stimulated the incorporation of both glucose and pyruvate in isolated cells and stimulated intercellular oxidation of glucose; in contrast, the lipolytic agents ACTH and isoproterenol actually inhibited the incorporation of pyruvate into adipocytes. In contrast to the effects of the lipolytic hormones, the effects of H3 and H4, like insulin, were not blocked by the presence of adenosine deaminase in the medium. The same concentrations of phenylarsine oxide were required to inhibit the stimulation of glucose incorporation whether by insulin or by histones. Furthermore, the addition of H4 or insulin to isolated adipocytes resulted in the increased phosphorylation of 17 kDa phosphoproteins as detected by two-dimensional electrophoresis. The insulin-like effect of the active histones was specific to their structure. Lysine-rich histones (H1, H2A and H2B), various polycations, and proteolytic fragments of purified H3 or H4 were all inactive. It is unknown whether this phenomenon might imply a physiological function for such endogenous molecules; however, a comparison of the detailed effects of insulin and histones might be informative in terms of common intracellular transduction systems.

Determination of monosaccharides in glycoproteins by reversed-phase high-performance liquid chromatography on 2.1-mm narrowbore columns.

The monosaccharide composition of glycoproteins was determined by using a Supelcosil LC-18 150 x 2.1 mm narrowbore column under reversed-phase conditions. Methodology was developed for sample hydrolysis, N-acetylation, benzoylation, and cleanup prior to high-performance liquid chromatography. Monosaccharides were identified and quantified at picomole levels. Various glycoproteins were then analyzed, including ribonucleases A and B, Mucin, and fetuin. In addition, the monosaccharide content of Chinese hamster ovary cell-expressed recombinant human renin was determined.

Separation of protein hormones

The purpose of this review is to highlight modern techniques in HPLC and electrophoresis used for protein hormone separations. The advent of biotechnological methods for production of synthetic polypeptides and recombinant proteins will have a significant future impact on the types of therapeutics and metabolites that need to be monitored in the clinical laboratory. The protein hormone examples given in this work were selected because of the comprehensive body of separation science literature and not necessarily for their future importance in medicine. The intention was to present an array of general methods and techniques which may be useful to the clinical investigator for analysis of any protein hormone.

Isolation and sequence analysis of proteins from mouse forebrain using two-dimensional gel electrophoresis coupled to high-pressure liquid extrusion

This report presents a technique for recovery of mouse forebrain proteins from two-dimensional sodium dodecyl sulfate-polyacrylamide gels for subsequent primary structure determination. Proteins were visualized by Coomassie staining or salt precipitation and manually cut out of the gel. Excised spots were minced and loaded into an empty precolumn of a reversed-phase high-performance liquid chromatography system. Purified protein was extruded from a gel matrix by pressurized liquid, then separated from gel contaminants by reversed-phase gradient elution, and finally collected in siliconized tubes or on polybrene-coated filter disks for gas-phase sequencing. Several mouse and rat forebrain proteins were purified by this method and sequenced. Three previously unidentified mouse brain proteins with molecular weights of 4,000, 12,000, and 18,500 were partially sequenced and three hemoglobin fragments were structurally identified and mapped. Ribonuclease A, myoglobin, adrenocorticotropin, and bovine somatotropin were also subjected to two-dimensional (2-D) analysis and partially sequenced. Recovery values of 27-95% were obtained for extruded 14C-labeled ribonuclease, carbonic anhydrase, and bovine serum albumin out of sodium dodecyl sulfate-polyacrylamide gel electrophoretic gels. Losses resulting from the multiple handling steps of a 2-D gel separation process were also investigated. Recoveries of 12-17%, as determined by sequencing signals, were achieved. These latter recovery values reflect overall losses incurred in gel-focusing, gel-sizing, staining, destaining, high-pressure liquid extrusion, and N-terminal blockage. This work demonstrates that an array of protein spots can be systematically identified or defined by partial sequencing after high-pressure liquid extrusion from a 2-D gel matrix.(ABSTRACT TRUNCATED AT 250 WORDS)