| 191. |
- Nazarian, Arpi, et al.
(författare)
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Inhibition of Circulating Dipeptidyl Peptidase 4 Activity in Patients with Metastatic Prostate Cancer
- 2014
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Ingår i: Molecular & Cellular Proteomics. - 1535-9484. ; 13:11, s. 3082-3096
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Tidskriftsartikel (refereegranskat)abstract
- Cancer is responsible for many deaths and is a major source of healthcare expenditures. The identification of new, non-invasive biomarkers might allow improvement of the direct diagnostic or prognostic ability of already available tools. Here, we took the innovative approach of interrogating the activity of exopeptidases in the serum of cancer patients with the aim of establishing a distinction based on enzymatic function, instead of simple protein levels, as a means to biomarker discovery. We first analyzed two well-characterized mouse models of prostate cancer, each with a distinct genetic lesion, and established that broad exopeptidase and targeted aminopeptidase activity tests reveal proteolytic changes associated with tumor development. We also describe new peptide-based freeze-frame reagents uniquely suited to probe the altered balance of selected aminopeptidases, as opposed to the full array of exopeptidases, and/or their modulators in patient serum or plasma. One particular proteolytic activity was impaired in animals with aggressive disease relative to cancer-free littermates. We identified the protease in question as dipeptidyl peptidase 4 (DPP4) by analyzing selected knockout mice and evaluating the effect of specific inhibitors. DPP4 activity was also reduced in the sera of patients with metastatic prostate cancer relative to patients with localized disease or healthy controls. However, no significant differences in DPP4 serum levels were observed, which established the loss of activity as the result of impaired enzymatic function. Biochemical analysis indicated that reduced activity was the result not of post-translational modifications or allosteric changes, but instead of a low-molecular-weight inhibitor. After we adjusted for age and total prostate-specific antigen, reduced DPP4 activity remained a significant predictor of cancer status. The results of this proof-of-principle study suggest that DPP4 activity might be a potential blood-based indicator of the presence of metastatic cancer of prostatic origin, either by itself or, more likely, as a means to improve the sensitivity and specificity of existing markers.
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| 192. |
- O'Connell, David, et al.
(författare)
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Integrated protein array screening and high throughput validation of 70 novel neural calmodulin binding proteins
- 2010
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Ingår i: Molecular & Cellular Proteomics. - 1535-9484. ; 9:6, s. 1118-1132
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Tidskriftsartikel (refereegranskat)abstract
- Calmodulin is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca(2+) ion concentration. To profile protein-protein interactions of calmodulin in human brain, we probed a high content human protein array with fluorophore-labelled calmodulin in the presence of Ca(2+). This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human neural proteins from a human brain cDNA library. We designed a screen to find high affinity (K(D) = 1 muM) binding partners of calmodulin and identified 76 human proteins from all intracellular compartments, of which 72 are novel. We measured the binding kinetics of 74 targets with calmodulin using a high throughput surface plasmon resonance assay. Most of the novel calmodulin-target complexes identified have low dissociation rates (koff = 10(3) s(-1)) and high affinity (K(D) = 1 muM), consistent with the design of the screen. Many of the identified proteins are known to assemble in neural tissue, forming assemblies such as the spectrin scaffold and the postsynaptic density. We have developed a microarray of the identified target proteins with which we can characterise the biochemistry of calmodulin for all targets in parallel. Four novel targets were verified in neural cells by co-immunoprecipitation, and four were selected for exploration of the calmodulin-binding regions. Using synthetic peptides and isothermal titration calorimetry, calmodulin binding motifs were identified in the potassium voltage gated channel Kv6.1, (residues 474-493), CaM kinase-like vesicle-associated protein (302-316), EF-hand domain family member A2 (202-216) and phosphatidylinositol-4-phosphate 5-kinase, type I, gamma (400-415).
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| 193. |
- Olsson, Niclas, et al.
(författare)
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Grading breast cancer tissues using molecular portraits.
- 2013
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Ingår i: Molecular & Cellular Proteomics. - 1535-9484. ; 12:12, s. 3612-3623
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Tidskriftsartikel (refereegranskat)abstract
- Tumor progression and prognosis of breast cancer patients is difficult to assess using current clinical and laboratory parameters, where a pathological grading is indicative of tumor aggressiveness. This grading is based on assessment of nuclear grade, tubule formation, and mitotic rate. We report here the first protein signatures associated with histological grades of breast cancer, using a novel affinity proteomics approach. We profiled 52 breast cancer tissue samples, by combining nine antibodies and label-free LC-MS/MS, which generated detailed quantified proteomic maps representing 1,388 proteins. The results showed that we could define in-depth molecular portraits of histologically graded breast cancer tumors. Consequently, a 49-plex candidate tissue protein signature was defined that discriminated between histological grade 1, 2, and 3 of breast cancer tumors with high accuracy. Highly biologically relevant proteins were identified, and the differentially expressed proteins indicated further support for the current hypothesis regarding remodeling of tumor microenvironment during tumor progression. The protein signature was corroborated using meta-analysis of transcriptional profiling data from an independent patient cohort. In addition, the potential for using the markers to estimate the risk of distant metastasis free survival was also indicated. Taken together, these molecular portraits could pave the way for improved classification and prognostication of breast cancer.
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| 194. |
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| 195. |
- Olsson, Niclas, et al.
(författare)
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Proteomic analysis and discovery using affinity proteomics and mass spectrometry.
- 2011
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Ingår i: Molecular & Cellular Proteomics. - 1535-9484. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Antibody-based microarrays are a rapidly evolving affinity-proteomic methodology that recently has shown great promise in clinical applications. The resolution of these proteomic analyses is, however, directly related to the number of data-points, i.e. antibodies, included on the array. Currently, this is a key bottleneck due to limited availability of numerous highly-characterized antibodies. Here, we present a conceptually new method, denoted global proteome survey, opening up the possibility to probe any proteome in a species independent manner while still using a limited set of antibodies. We use context-independent-motif-specific antibodies directed against short amino acid motifs, where each motif is present in up to a few hundred different proteins. First, the digested proteome is exposed to these antibodies, whereby motif-containing peptides are enriched, which then are detected and identified by mass spectrometry. In this study, we profiled extracts from human colon tissue, yeast cells lysate, and mouse liver tissue to demonstrate proof-of-concept.
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| 196. |
- Olsson, Niclas, et al.
(författare)
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Quantitative proteomics targeting classes of motif-containing peptides using immunoaffinity-based mass spectrometry.
- 2012
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Ingår i: Molecular & Cellular Proteomics. - 1535-9484. ; 11:8, s. 342-354
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Tidskriftsartikel (refereegranskat)abstract
- The development of high-performance technology platforms for generating detailed protein expression profiles, or protein atlases, is essential. Recently, we presented a novel platform that we termed global proteome survey (GPS), where we combined the best features of affinity proteomics and mass spectrometry, to probe any proteome in a species independent manner while still using a limited set of antibodies. We used so called context-independent-motif-specific antibodies, directed against short amino acid motifs. This enabled enrichment of motif-containing peptides from a digested proteome, which then were detected and identified by mass spectrometry. In this study, we have demonstrated the quantitative capability, reproducibility, sensitivity, and coverage of the GPS technology by targeting SILAC-labeled yeast cultures cultivated in glucose or ethanol. The data showed that a wide range of motif-containing peptides (proteins) could be detected, identified, and quantified in a highly reproducible manner. On average, each of six different motif-specific antibodies was found to target about 75 different motif-containing proteins. Furthermore, peptides originating from proteins spanning in abundance from over a million down to less than 50 copies per cell, could be targeted. It is worth noting that a significant set of peptides previously not reported in the PeptideAtlas database was among the profiled targets. The quantitative data corroborated well with the corresponding data generated after conventional SCX fractionation of the same samples. Finally, several differentially expressed proteins, with both known and unknown functions, many relevant for the central carbon metabolism, could be detected in the glucose- versus ethanol-cultivated yeast. Taken together, the study demonstrated the potential of our immunoaffinity-based mass spectrometry platform for reproducible quantitative proteomics targeting classes of motif-containing peptides.
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| 197. |
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| 198. |
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| 199. |
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| 200. |
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