An 8-plex version of an isobaric reagent for the quantitation of proteins using shotgun methods is presented. The 8-plex version of the reagent relies on amine-labeling chemistry of peptides similar to 4-plex reagents. MS/MS reporter ions at 113, 114, 115, 116, 117, 118, 119, and 121 m/z are used to quantify protein expression. This technology which was first applied to a test mixture consisting of eight proteins and resulted in accurate quantitation, has the potential to increase throughput of analysis for quantitative shotgun proteomics experiments when compared to 2- and 4-plex methods. The technology was subsequently applied to a longitudinal study of cerebrospinal fluid (CSF) proteins from subjects undergoing intravenous Ig treatment for Alzheimer's disease. Results from this study identify a number of protein expression changes that occur in CSF after 3 and 6 months of treatment compared to a baseline and compared to a drug washout period. A visualization tool was developed for this dataset and is presented. The tool can aid in the identification of key peptides and measurements. One conclusion aided by the visualization tool is that there are differences in considering peptide-based observations versus protein-based observations from quantitative shotgun proteomics studies.

Downstream processing of monoclonal antibodies (mAbs) has evolved to allow the specific process for a new product to be developed largely by empirical specialization of a platform process that enables removal of impurities of different kinds. A more complete characterization of impurities and the product itself would provide insights into the rational design of efficient downstream processes. This work identifies and characterizes host cell protein (HCP) product associated impurities, i.e., HCP species carried through the downstream processes via direct interactions with the mAb. Interactions between HCP and mAbs are characterized using cross interaction chromatography under solution conditions typical of those used in downstream processing. The interacting species are then identified by two dimensional gel electrophoresis and mass spectrometry. This methodology has been applied to identify product associated impurities in one particular purification step, namely protein A affinity chromatography, for four therapeutic mAbs as well as the Fab and Fc domains of one of these mAbs. The results show both the differences in HCP-mAb interactions among different mAbs, and the relative importance of product association compared to co-elution in protein A affinity chromatography.

Shotgun proteomic methods involving isobaric tagging of peptides enable high-throughput proteomic analysis. iTRAQ reagents allow simultaneous identification and quantitation of proteins in four different samples using tandem mass spectrometry (MS). In this article, we provide a brief description of proteome analysis using iTRAQ reagents and review the current applications of these reagents in proteomic studies. We also compare different aspects of protein identification including protein sequence coverage and proteome coverage obtained using iTRAQ reagents with those using other shotgun proteomic techniques. We briefly discuss the issue of isotope purity correction in measured peak areas during protein quantitation using iTRAQ reagents. Finally, we conclude with some of the current challenges in MS-based proteomic analysis that are limiting protein identifications obtained by different shotgun proteomic methods.

A panel of possible CSF biomarkers for AD has been identified using proteomic methods.

A comparison of the consistency of proteome quantitation using two-dimensional electrophoresis and shotgun isobaric tagging in Escherichia coli cellsAn important consideration in the measurement of quantitative changes in protein expression is the consistency of the observations for a given technique as well as the reproducibility of the experiment. A quantitative assessment of the technical and biological variability is crucial to avoid erroneous inferences and conclusions. Two methods for measuring quantitative changes in protein expression are two-dimensional electrophoresis (2-DE) and shotgun proteomics of isobaric-tagged samples using iTRAQ  reagents. An assessment of changes in Escherichia coli protein expression in response to rhsA induction demonstrates that half of the quantified protein expression ratios have a coefficent of variation (CV) less than 0.31 using 2-DE and less than 0.24 using isobaric tags; whereas 95% of the quantified protein expression ratios have a CV less than 0.81 using 2-DE and less than 0.53 using isobaric tags. The selective removal of outlier data points from the shotgun method using Grubb's and Rosner's statistical outlier tests improves the consistency of the quantitation data obtained.

Microbial communities are of great environmental, medical, and industrial significance. To date, biomolecular methods to study communities have focused on identifying species, with limited capabilities to reveal functions. Proteomics has the potential to yield functional information about these communities, but the application of proteomic methods to complex mixtures of unsequenced organisms is in its infancy. In this study, 2DE, MALDI-TOF/TOF MS, and de novo peptide sequencing were used for the separation and identification of proteins differentially expressed over time following exposure of a bacterial community to an inhibitory level of cadmium. Significant community proteome responses after 0.25, 1, 2, and 3 h of exposure to cadmium were observed, with more than 100 protein expression changes detected at each time point. Several temporal responses were observed, and the most common expression pattern was immediate up- or down-regulation within 15 min of shock followed by maintenance of that level. More than 100 unique differentially expressed proteins were identified through database searching and de novo sequencing. Proteins of importance in the cadmium shock included ATPases, oxidoreductases, and transport proteins. The ability of proteomics to detect the differential regulation of these proteins even during short cadmium exposures shows that it is a powerful tool in explaining cellular mechanisms for a mixed culture. This is the first report of the large-scale identification of proteins involved in the dynamic response of a community of unsequenced bacteria using de novo sequencing.

Bovine pericardium is an important biomaterial with current application in glutaraldehyde-fixed bioprosthetic heart valves and possible future application as an unfixed biological scaffold for tissue engineering. The importance of both humoral and cell-mediated rejection responses toward fixed and unfixed xenogeneic tissues has become increasingly apparent. However, the full scope and specific identities of bovine pericardium proteins that can elicit an immune response remain largely unknown. In this study, an immunoproteomic approach was used to survey bovine pericardium proteins for their ability to elicit a humoral immune response in rabbits. A two-stage protein extraction protocol was used to separate bovine pericardium proteins into water-and lipid-soluble fractions. Two-dimensional gel electrophoresis was performed to separate the proteins from each fraction. Western blots were generated from two-dimensional gels of both bovine pericardium protein fractions. These blots were probed with serum from rabbits immunized with bovine pericardium and a secondary antibody was used to assess for IgG positivity. Western blots were compared to duplicate two-dimensional gels and proteins in matched spots were identified by tandem mass spectrometry. Thirty-one putative protein antigens were identified, eight of which are known to be antigenic from previous studies. All of the putative antigens demonstrated progressive staining intensity with increasing days of post-exposure serum. Identified antigenic proteins represented a variety of functional and structural protein types, and included both cellular and matrix proteins. The results of this study have implications for the use of bovine pericardium as a biomaterial in bioprostheses and tissue engineering applications, as well as xenotransplantation in general.

We describe the use of amine-specific isobaric tags for protein expression quantification to study the effect of rhsA element over-expression in Escherichia coli. The use of an isobaric tagging strategy facilitates a shotgun approach to proteomic analysis and enables quantitation of up to four samples in parallel, based on the reporter ion series using tandem mass spectrometry (MS/MS). Using a liquid chromatography matrix-assisted laser desorption/ionization approach, 23,139 MS/MS spectra were collected. Five thousand sixty-three peptides derived from 780 proteins were quantified including several lower abundance proteins, such as transcription factors, DnaB and DnaG. More than 65% of the proteins had at least two high confidence peptide matches per protein (p<0.05). Further, a statistical test based on the Grubb's and Rosner's tests was able to discriminate outlier data. The removal of outlier data had no significant effect on the functional categories of proteins that were represented in the study.