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 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 ...

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.

An unbiased survey has been made of the stable, most abundant multi-protein complexes in Desulfovibrio vulgaris Hildenborough (DvH) that are larger than Mr Ϸ 400 k. The quaternary structures for 8 of the 16 complexes purified during this work were determined by single-particle reconstruction of negatively stained specimens, a success rate Ϸ10 times greater than that of previous ''proteomic'' screens. In addition, the subunit compositions and stoichiometries of the remaining complexes were determined by biochemical methods. Our data show that the structures of only two of these large complexes, out of the 13 in this set that have recognizable functions, can be modeled with confidence based on the structures of known homologs. These results indicate that there is significantly greater variability in the way that homologous prokaryotic macromolecular complexes are assembled than has generally been appreciated. As a consequence, we suggest that relying solely on previously determined quaternary structures for homologous proteins may not be sufficient to properly understand their role in another cell of interest.comparative evolutionary analysis ͉ single-particle electron microscopy ͉ structural homology

Quantifying the variation in the human plasma proteome is an essential prerequisite for disease-specific biomarker detection. We report here on the longitudinal and individual variation in human plasma characterized by two-dimensional difference gel electrophoresis (2-D DIGE) using plasma samples from eleven healthy subjects collected three times over a two week period. Fixed-effects modeling was used to remove dye and gel variability. Mixed-effects modeling was then used to quantitate the sources of proteomic variation. The subject-to-subject variation represented the largest variance component, while the time-within-subject variation was comparable to the experimental variation found in a previous technical variability study where one human plasma sample was processed eight times in parallel and each was then analyzed by 2-D DIGE in triplicate. Here, 21 protein spots had larger than 50% CV, suggesting that these proteins may not be appropriate as biomarkers and should be carefully scrutinized in future studies. Seventy-eight protein spots showing differential protein levels between different individuals or individual collections were identified by mass spectrometry and further characterized using hierarchical clustering. The results present a first step toward understanding the complexity of longitudinal and individual variation in the human plasma proteome, and provide a baseline for improved biomarker discovery.