Absolute quantitation of cancer-related proteins using an MS-based peptide chip

New technologies are needed that can diagnose cancer more rapidly and accurately. These technologies must also have the ability to identify the particular cellular abnormalities contributing to the malignancy, thus directing the appropriate treatments. Such technologies should permit absolute quantitation of specific tumor biomarkers and their level of posttranslational modifications. Quantitative molecular profiling of cancer signaling networks would provide a more detailed understanding of the contribution of protein expression and posttranslational modification levels to tumorigenesis. We have developed a unique approach for absolute quantitation of protein expression that integrates affinity capture of proteolytic peptides with mass spectrometry and thus provides detection, identification, and quantitation of their cognate proteins. We have previously shown the high sensitivity and specificity of this approach. Here we demonstrate the absolute quantitation of a model peptide using our technology. We have used this approach to capture epitope-containing peptides from proteolytically digested target proteins, including p53, epidermal growth factor receptor (EGFR), and prostate-specific antigen (PSA). Our technology can easily be extended to the absolute quantitation of protein modification levels, in addition to the determination of protein expression levels, and can be readily adapted for use in a microarray format. This method offers an improved approach to protein chip technology that should prove useful for clinical diagnosis and drug development applications.     

Adipose tissue is a regulated source of interleukin-10

White adipose tissue (WAT) is the source of pro- and anti-inflammatory cytokines and we have recently shown that this tissue is a major source of the anti-inflammatory interleukin (IL)-1 receptor antagonist (IL-1Ra). We now aimed at identifying additional adipose-derived cytokines, which might serve as regulators of IL-1Ra. We demonstrate here for the first time that the antiinflammatory cytokine IL-10 is secreted by human WAT explants and that it is up-regulated by LPS and TNF-alpha in vitro, as well as in obesity in humans (2- and 6-fold increase in subcutaneous and visceral WAT, respectively) and rodents (4-fold increase).     

TGF-beta1 codon 25 gene polymorphism is associated with cirrhosis in patients with hereditary hemochromatosis

Hereditary hemochromatosis (HHC) is an autosomal recessive disorder of iron metabolism with variable penetrance. Only a minority of C282Y homozygotes develop clinical overt disease and cirrhosis. The phenotypic heterogeneity of HHC may be due to host genetic factors influencing fibrogenesis such as cytokine gene polymorphisms. In this respect, we investigated the impact of functional genetic polymorphisms of TGF-beta1 (codon 10 Leu/Pro, codon 25 Arg/Pro), TNF-alpha (-308 G/A, -238 G/A) and angiotensinogen (-6 G/A) on the development of cirrhosis in HHC. One hundred and forty-nine (111 male, mean age: 51.0+/-12.9) C282Y homozygotes who underwent liver biopsy were studied. Genotyping was performed by RFLP analysis. TGF-beta1 codon 25 genotypes Arg/Pro and Pro/Pro were more common in patients with cirrhosis than in those without (23.6% vs. 7.4%, p = 0.005). In contrast, the distribution of TGF-beta1 codon 10, TNF-alpha and angiotensinogen genotypes was not different. Logistic regression analysis identified male sex, age, serum ferritin and TGF-beta1 codon 25 Arg/Pro and Pro/Pro as independent predictors for the presence of cirrhosis. The adjusted odds ratio for TGF-beta1 codon 25 Arg/Pro and Pro/Pro was 2.8 (95% CI 1.4-5.7, p = 0.004). In conclusion, C282Y homozygotes carrying TGF-beta1 genotypes Arg/Pro and Pro/Pro are more likely to develop cirrhosis than those with genotype Arg/Arg.     

Potential approaches to prevent uncommon hemolytic side effects of AB0 antibodies in plasma derivatives.

Since hemolytic reactions in patients after administration of plasma derivatives like immunoglobulins or coagulation factor preparations have been described, titers of anti-A and anti-B-antibodies have to be below defined levels for batch release of these plasma-derived therapeutic products according to the European Pharmacopoeia. We have summarized clinical relevance of AB0 antibodies in plasma derivatives and related legal issues in the European Union, United States of America, and Japan. We have also discussed potential approaches for the prevention of hemolytic side effects with feasible steps in preparation of plasma derivatives, viz., (1) selection of donors, (2) exclusion of "dangerous donors", (3) optimizing ratio of the types of plasma, (4) removal of antibodies, (5) production of blood-group-specific plasma derivatives, (6) rejection of batches of plasma derivatives with high titers of antibodies, and (7) crossmatching before administration. For harmonization of standards for anti-A and anti-B in plasma-derived therapeutics the regulators and manufacturers will have to realistically deal with complex clinical, practical, and economic issues.     

Utilizing the intramolecular Fukuyama-Mitsunobu reaction for a flexible synthesis of novel heterocyclic scaffolds for peptidomimetic drug design

We report the synthesis of the novel scaffolds pyrazino[1,2-b]isoquinoline and pyrrolo[1,2-a]pyrazine displaying the somatostatin pharmacophores. Both classes of compounds contain a pyrazine heterocycle, which can be prepared in a straightforward manner utilizing an intramolecular Fukuyama-Mitsunobu reaction. As both the families derive from amino acids, they can be accessed in high optical purity.     

Overexpression of cathepsin B in gastric cancer identified by proteome analysis

We aimed to validate an analytical approach based on proteomics on gastric cancer specimens for the identification of new putative diagnostic or prognostic markers. Primary screening was performed on gastrectomy specimens obtained from ten consecutive patients with gastric cancer. Gastric epithelial cells were obtained with an epithelial cell enrichment technique, homogenized and then separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The differential protein expression pattern was verified stepwise by Western blotting and immunohistochemistry on samples from 28 and 46 cancer patients, respectively. The putative clinical applicability and prognostic use were tested by an enzyme-linked immunoabsorbent assay on serum samples obtained from 149 cancer patients. One hundred-ninety-one differentially expressed protein spots were found by 2-D PAGE and identified by mass spectrometry, including cathepsin B, which was over-expressed in six (60%) patients. Western blotting confirmed that the active form of cathepsin B is over-expressed, while immunohistochemistry showed strong cytoplasmic staining in cancer tissues of 45 (98%) patients. The serum level of cathepsin B was increased in patients with gastric cancer compared to healthy controls (P = 0.0026) and correlated with T-category and the presence of distant metastases (P < 0.05). Serum levels above 129 pmol x L(-1) were associated with a reduced survival rate (P = 0.0297). Proteome analysis is a valuable tool for the identification of prognostic markers in gastric cancer: Increased cathepsin B serum levels are associated with advanced tumor stages and progressive disease, which enables the classification of some gastric cancer patients into a subgroup that should undergo aggressive therapy.     

Peptidomic analysis of human blood specimens: comparison between plasma specimens and serum by differential peptide display

The human Plasma Proteome Project pilot phase aims to analyze serum and plasma specimens to elucidate specimen characteristics by various proteomic techniques to ensure sufficient sample quality for the HUPO main phase. We used our proprietary peptidomics technologies to analyze the samples distributed by HUPO. Peptidomics summarizes technologies for visualization, quantitation, and identification of the low-molecular-weight proteome (<15 kDa), the "peptidome." We analyzed all four HUPO specimens (EDTA plasma, citrate plasma, heparin plasma, and serum) from African- and Asian-American donors and compared them to in-house collected Caucasian specimens. One main finding focuses on the most suitable method of plasma specimen collection. Gentle platelet removal from plasma samples is beneficial for improved specificity. Platelet contamination or activation of platelets by low temperature prior to their removal leads to distinct and multiple peptide signals in plasma samples. Two different specimen collection protocols for platelet-poor plasma are recommended. Further emphasis is placed on the differences between plasma and serum on a peptidomic level. A large number of peptides, many of them in rather high abundance, are only present in serum and not detectable in plasma. This ex vivo generation of multiple peptides hampers discovery efforts and is caused by a variety of factors: the release of platelet-derived peptides, other peptides derived from cellular components or the clot, enzymatic activities of coagulation cascades, and other proteases. We conclude that specimen collection is a crucial step for successful peptide biomarker discovery in human blood samples. For analysis of the low-molecular-weight proteome, we recommend the use of platelet-depleted EDTA or citrate plasma.     

MtdC, a novel class of methylene tetrahydromethanopterin dehydrogenases

Novel methylene tetrahydromethanopterin (H4MPT) dehydrogenase enzymes, named MtdC, were purified after expressing in Escherichia coli genes from, respectively, Gemmata sp. strain Wa1-1 and environmental DNA originating from unidentified microbial species. The MtdC enzymes were shown to possess high affinities for methylene-H4MPT and NADP but low affinities for methylene tetrahydrofolate or NAD. The substrate range and the kinetic properties revealed by MtdC enzymes distinguish them from the previously characterized bacterial methylene-H4MPT dehydrogenases, MtdA and MtdB. While revealing higher sequence similarity to MtdA enzymes, MtdC enzymes appear to fulfill a function homologous to the function of MtdB, as part of the H4MPT-linked pathway for formaldehyde oxidation/detoxification.