Two-dimensional differential gel electrophoresis (2-D DIGE) was used to analyze human serum following the removal of albumin and five other high-abundant serum proteins. After protein removal, serum was analyzed by SDS-PAGE as a preliminary screen, and significant differences between four high-abundant protein removal methods were observed. Antibody-based albumin removal and high-abundant protein removal methods were found to be efficient and specific. To further characterize serum after protein removal, 2-D DIGE was employed, enabling multiplexed analysis of serum through the use of three fluorescent protein dyes. Comparison between crude serum and serum after removal of high-abundant proteins clearly illustrates an increase in the number of lower abundant protein spots observed. Approximately 850 protein spots were detected in crude serum whereas over 1500 protein spots were exposed following removal of six high-abundant proteins, representing a 76% increase in protein spot detection. Several proteins that showed a 2-fold increase in intensity after depletion of high-abundant proteins, as well as proteins that were depleted during abundant protein removal methods, were further characterized by mass spectrometry. This series of experiments demonstrates that high-abundant protein removal, combined with 2-D DIGE, is a practical approach for enriching and characterizing lower abundant proteins in human serum. Consequently, this methodology offers advances in proteomic characterization, and therefore, in the identification of biomarkers from human serum.

The primary structure of PI31, a protein inhibitor of the 20 S proteasome, was deduced by cDNA cloning and sequencing. The human protein has a calculated molecular weight of 29,792, a value in excellent accord with 31,000, as estimated by SDS-polyacrylamide gel electrophoresis for purified bovine PI31, and is not similar to any other protein in current data bases. PI31 is a proline-rich protein, particularly within its carboxyl-terminal half where 26% of the amino acids are proline. Wildtype PI31 and various truncation mutants were expressed in Escherichia coli and purified to homogeneity. Recombinant wild-type PI31 displayed structural and functional properties similar to those of PI31 purified from bovine red blood cells and inhibited the hydrolysis of protein and peptide substrates by the 20 S proteasome. Analysis of truncation mutants demonstrated that proteasome inhibition was conferred by the carboxyl-terminal proline-rich domain of PI31, which appears to have an extended secondary structure. Inhibition of the 20 S proteasome by PI31 involved formation a proteasome-PI31 complex. In addition to its direct inhibition of the 20 S proteasome, PI31 inhibited the activation of the proteasome by each of two proteasome regulatory proteins, PA700 and PA28. These results suggest that PI31 plays an important role in control of proteasome function, including that in ubiquitin-dependent pathways of protein degradation.

Previously, it was shown that optimal functioning of the Yersinia type III secretion system (T3SS) in cell culture infection assays requires the exoribonuclease polynucleotide phosphorylase (PNPase) and that normal T3SS activity could be restored in the Deltapnp strains by expressing just the approximately 70-aa S1 RNA-binding domain of PNPase. Here, it is shown that the Yersinia Deltapnp strain is less virulent in the mouse compared with the isogenic wild-type strain. To begin to understand what could be limiting T3SS activity in the absence of PNPase, T3SS-encoding transcripts and proteins in the YersiniaDeltapnp strains were analyzed. Surprisingly, it was found that the Deltapnp Yersinia strains possessed enhanced levels of T3SS-encoding transcripts and proteins compared with the wild-type strains. We then found that an S1 variant containing a disruption in its RNA-binding subdomain was inactive in terms of restoring normal T3SS activity. However, T3SS expression levels did not differ between Deltapnp strains expressing active and inactive S1 proteins, further showing that T3SS activity and expression levels, at least as related to PNPase and its S1 domain, are not linked. The results suggest that PNPase affects the expression and activity of the T3SS by distinct mechanisms and that the S1-dependent effect on T3SS activity involves an RNA intermediate.

The Yersinia pestis proteome was studied as a function of temperature and calcium by two-dimensional differential gel electrophoresis. Over 4,100 individual protein spots were detected, of which hundreds were differentially expressed. A total of 43 differentially expressed protein spots, representing 24 unique proteins, were identified by mass spectrometry. Differences in expression were observed for several virulence-associated factors, including catalase-peroxidase (KatY), murine toxin (Ymt), plasminogen activator (Pla), and F1 capsule antigen (Caf1), as well as several putative virulence factors and membrane-bound and metabolic proteins. Differentially expressed proteins not previously reported to contribute to virulence are candidates for more detailed mechanistic studies, representing potential new virulence determinants.Yersinia pestis, the etiological agent of plague, is a gram-negative bacterium that is both a natural environmental pathogen and a biothreat agent (4,8,32). Early studies of Yersinia physiology uncovered the low calcium response (LCR), whereby bacterial cultures grown in rich medium at an elevated temperature (37°C) exhibit a growth defect upon chelation of calcium ions. The growth arrest was shown to be a result of one of the two type III secretion systems (TTSSs) in Y. pestis, the Ysc TTSS, and is responsible for the secretion of virulence factors known as Yersinia outer proteins, or Yops (21, 29; for a review, see reference 61). This TTSS can be activated in vitro and virulence factors can be released into the medium when Y. pestis is grown at 37°C with submillimolar calcium (for a review, see reference 16). Upon interaction with the host, the TTSS enables virulence factors to enter the host cell through a specialized apparatus, the injectisome (15). Once inside the host cell, Yops affect a variety of host pathways, with detectable expression changes in the pathogen as well as the host (14,52,82).The Y. pestis proteome was previously examined using twodimensional electrophoresis (57,60,71,72). These studies demonstrated that virulence factors were not induced at 26°C or 37°C in the presence of calcium concentrations similar to that found in mammalian plasma (2.5 mM) (71). More recently, the introduction of two-dimensional differential gel electrophoresis (2-D DIGE) has significantly improved the quality of gel-based proteomics through fluorescence-based multiplex analyses providing relative quantitation of expression differences and improved gel-to-gel comparisons (75). Several examples of 2-D DIGE bacterial proteomics have been reported (23), including characterizations of the gram-negative bacterium Escherichia coli (1, 76, 81). Here we report the characterization of the soluble cell-associated proteome of Y. pestis as a function of temperature and calcium, which were used to effect virulence induction. Differentially expressed proteins include virulence-associated factors, membrane-bound proteins, metabolic and housekeeping proteins, and potential new virulence determinants.Bacterial ...

Application of the more stringent statistical tests applied to the normalized 2D DIGE data decreased the number of potentially differentially expressed proteins from the number obtained from DeCyder and increased the confidence in detecting differential expression in human clinical studies.

The complexity of human plasma presents a number of challenges to the efficient and reproducible proteomic analysis of differential expression in response to disease. Before individual variation and disease-specific protein biomarkers can be identified from human plasma, the experimental variability inherent in the protein separation and detection techniques must be quantified. We report on the variation found in two-dimensional difference gel electrophoresis (2-D DIGE) analysis of human plasma. Eight aliquots of a human plasma sample were subjected to top-6 highest abundant protein depletion and were subsequently analyzed in triplicate for a total of 24 DIGE samples on 12 gels. Spot-wise standard deviation estimates indicated that fold changes greater than 2 can be detected with a manageable number of replicates in simple ANOVA experiments with human plasma. Mixed-effects statistical modeling quantified the effect of the dyes, and segregated the spot-wise variance into components of sample preparation, gel-to-gel differences, and random error. The gel-to-gel component was found to be the largest source of variation, followed by the sample preparation step. An improved protocol for the depletion of the top-6 high-abundance proteins is suggested, which, along with the use of statistical modeling and future improvements in gel quality and image processing, can further reduce the variation and increase the efficiency of 2-D DIGE proteomic analysis of human plasma.

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The accurate mass and time tag mass spectrometry method and clustering analysis were used to compare the abundance change of 992 Yersinia pestis proteins under four contrasting growth conditions (26 and 37 degrees C, with or without Ca2+) that mimicked growth states in either a flea vector or mammalian host. Eighty-nine proteins were observed to have similar abundance change profiles to 29 known virulence associated proteins, providing identification of additional biomarker candidates. Eighty-seven hypothetical proteins, which clustered into 5 distinct clusters of like-protein abundance change, were identified as unique biomarkers related specifically to growth condition.