Automated assignment of ionization states in broad-mass matrix-assisted laser desorption/ionization spectra of protein mixtures.

A computational technique is presented for the automated assignment of the multiple charge and multimer states (ionization states) in the time-of-flight (TOF) domain for matrix-assisted laser desorption/ionization (MALDI) spectra. Examples of the application of this technique include an improved, automatic calibration over the 2 to 70 kDa mass range and a reduced data redundancy after reconstruction of the molecular spectrum of only singly charged monomers. This method builds on our previously reported enhancement of broad-mass signal detection, and includes two steps: (1) an automated correction of the instrumental acquisition initial time delay, and (2) a recursive TOF detection of multiple charge states and singly charged multimers of molecular [MH](+) ions over the entire record range, based on MALDI methods. The technique is tested using calibration mixtures and pooled serum quality control samples acquired along with clinical study data. The described automated procedure improves the analysis and dimension reduction of MS data for comparative proteomics applications.

Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes.

Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr), an alkaline solution (180 mM Na2CO3, pH 11.3) and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14). Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i) integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries); (ii) peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries); (iii) cytoplasmic or ribosomal membrane-contaminating proteins (80 entries). Thirty-one proteins were experimentally associated with the outer membrane (OM). Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM) proteins with two or more predicted transmembrane domains (TMDs) were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six beta-barrel OM protein families and 25 distinct IM transporter families.

Characterizing the dynamic nature of the Yersinia pestis periplasmic proteome in response to nutrient exhaustion and temperature change.

The periplasmic proteome of Yersinia pestis strain KIM6+ was characterized using differential 2-DE display of proteins isolated from several subcellular fractions. Circa 160 proteins were designated as periplasmic, including 62 (putative) solute-binding proteins of ATP-binding cassette (ABC) transporters (SBPs) and 46 (putative) metabolic enzymes. More than 30 SBPs were significantly increased in abundance during stationary phase cell growth, compared to the exponential phase. The data suggest that nutrient exhaustion in the stationary phase triggers cellular responses resulting in the induced expression of numerous ABC transporters, which are responsible for the import of solutes/nutrients. Limited availability of inorganic phosphate (P(i)) also caused dramatic proteomic changes. Nine proteins were functionally linked to the mobilization and import of three small molecules (P(i), phosphonate and glycerol-3-phosphate) and accounted for nearly half of the total protein mass in the periplasm of P(i)-starved cells. When cells were grown at 26 degrees C versus 37 degrees C, corresponding to ambient temperatures in the flea vector and mammalian hosts, respectively, several periplasmic proteins with no known roles in the Y. pestis life cycle were strongly altered in abundance. This included a putative nitrate/sulfonate/bicarbonate-specific SBP (Y1004), encoded by the virulence-associated plasmid pMT1 and increased in abundance at 37 degrees C.

Optimization of MALDI-TOF MS detection for enhanced sensitivity of affinity-captured proteins spanning a 100 kDa mass range.

Analysis of complex biological samples by MALDI-TOF mass spectrometry has been generally limited to the detection of low-mass protein (or protein fragment) peaks. We have extended the mass range of MALDI-TOF high-sensitivity detection by an order of magnitude through the combined optimization of instrument parameters, data processing, and sample preparation procedures for affinity capture. WCX, C3, and IMAC magnetic beads were determined to be complementary and most favorable for broad mass range protein profiling. Key instrument parameters for extending mass range included adjustment of the ADC offset and preamplifier filter values of the TOF detector. Data processing was improved by a combination of constant and quadratic down-sampling, preceded by exponential baseline subtraction, to increase sensitivity of signal peaks. This enhancement in broad mass range detection of protein signals will be of direct benefit in MS expression profiling studies requiring full linear range mass detection.

Comparative proteomic analysis of Staphylococcus aureus strains with differences in resistance to the cell wall-targeting antibiotic vancomycin.

Three isogenic strains derived from a clinical vancomycin-intermediate Staphylococcus aureus isolate were examined by comparative protein abundance analysis. Subcellular fractionation was followed by protein separation in 2-DE gels and spot identification by MALDI-TOFTOF-MS and LC-MS/MS. Sixty-five significant protein abundance changes were determined. Numerous enzymes participating in the purine biosynthesis pathway were dramatically increased in abundance in strain VP32, which featured the highest minimal inhibitory concentration for vancomycin, compared to strains P100 and HIP5827. Peptidoglycan hydrolase LytM (LytM) and the SceD protein, a putative transglycosylase, were increased in abundance in the cell envelope fraction of strain VP32, whereas the enzyme D-Ala-D-Ala ligase was decreased in its cytosol fraction. Furthermore, penicillin-binding protein 2 (PBP2) had substantially higher activity in strain VP32 compared to that in strain HIP5827. LytM, PBP2 and D-Ala-D-Ala ligase catalyze reactions in the biosynthesis or the metabolism of cell wall peptidoglycan. It is plausible that expression and activity changes of these enzymes in strain VP32 are responsible for an altered cell wall turnover rate, which has been observed, and an altered peptidoglycan structure, which has yet to be elucidated for this highly vancomycin-resistant strain.