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- Végvári, Ákos, et al.
(författare)
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Bioinformatic strategies for unambiguous identification of prostate specific antigen in clinical samples
- 2011
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919. ; 75:1, s. 202-210
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Tidskriftsartikel (refereegranskat)abstract
- Prostate specific antigen (PSA), as a widely used clinical biomarker in prostate cancer diagnostics, exists in multiple molecular forms. However, all of these forms might not be recognized in a given sample by the standard immunoassays. Therefore, we have investigated PSA isoforms separated by size using mass spectrometric analyses. The objective of these developments was to identify and specify the various forms of PSA. To optimize successful identification of different PSA forms, we have developed a bioinformatic strategy, consisting of high resolution MALDI-MS PMF and sequencing MS/MS data searches. To improve sequence-based identification, the recently introduced Proteios software environment was employed, allowing the combination of multiple database search engines in an automated manner. We could unambiguously identify PSA in clinical samples by all detectable tryptic peptides, which were found to be common in several isoforms.
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| 3. |
- Végvári, Ákos, et al.
(författare)
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Identification of a Novel Proteoform of Prostate Specific Antigen (SNP-L132I) in Clinical Samples by Multiple Reaction Monitoring.
- 2013
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Ingår i: Molecular & Cellular Proteomics. - 1535-9484. ; 12:10, s. 2761-2773
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Tidskriftsartikel (refereegranskat)abstract
- Prostate specific antigen (PSA) is a well-established tumor marker, which is frequently employed as model biomarker to develop and evaluate emerging quantitative proteomics techniques, partially due to wide access to commercialized immunoassays serving as "gold standard". We designed a multiple reaction monitoring (MRM) assay to detect PSA proteoforms in clinical samples (n=72), utilizing specificity and sensitivity of the method. We report for the first time, a PSA proteoform, coded by SNP-L132I (rs2003783), observed in 9 samples in both heterozygous (n=7) and homozygous (n=2) expression profiles. Other isoforms of PSA, derived from protein databases, were not identified by four unique proteotypic tryptic peptides. We have also utilized our MRM assay for precise quantitative analysis of PSA concentrations in both seminal and blood plasma samples. The analytical performance was evaluated, providing close agreement between each quantitation based on three selected peptides (LSEPAELTDAVK, IVGGWECEK and SVILLGR) and a routinely used commercialized immunoassay. Additionally, we have disclosed that the peptide IVGGWECEK is shared with kallikrein-related peptidase 2 and therefore not unique for PSA. Hence, we propose to use another tryptic sequence (SVILLGR) for accurate MRM-quantification of PSA in clinical samples.
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| 4. |
- Végvári, Ákos, et al.
(författare)
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Identification of prostate specific antigen (PSA) isoforms in complex biological samples utilizing complementary platforms
- 2010
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919. ; 73:6, s. 1137-1147
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Tidskriftsartikel (refereegranskat)abstract
- Measurements of the prostate-specific antigen (PSA) levels in blood are widely used as diagnostic, predictive and prognostic marker of prostate disease. The selective detection of molecular forms of PSA can contribute clinically to meaningful enhancements of the conventional PSA-test. As it is plausible that an in-depth search for structural variants of PSA gene products may increase our ability to discriminate distinct patho-biological basis and stages of prostate diseases, we have developed a multi-step protocol comprising gel-based methods followed by mass spectrometric identification. Our current aim was to provide a comprehensive identification of PSA variants occurring in seminal fluid. We provide a proof-of-principle for this multiple step analytical approach to identify multiple PSA variants from complex biological samples that revealed distinct molecular characteristics. In addition, sequence-annotated protein bands in SDS–PAGE gels were compared to those detected by Western blots, and by monitoring the enzymatic activity in zymogram gels, using gelatin as a substrate. The high accuracy annotations were obtained by fast turnaround MALDI-Orbitrap analysis from excised and digested gel bands. Multiple PSA forms were identified utilizing a combination of MASCOT and SEQUEST search engines.
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| 5. |
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| 6. |
- Végvári, Ákos, et al.
(författare)
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Molecular microheterogeneity of prostate specific antigen in seminal fluid by mass spectrometry
- 2012
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Ingår i: Clinical Biochemistry. - : Elsevier BV. - 0009-9120 .- 1873-2933. ; 45:4-5, s. 331-338
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: Prostate specific antigen (PSA) is a widely used and clinically valuable marker for prostate disease. In order to enable the development of new PSA assays and progress the understanding of the biology of PSA we have analyzed PSA in seminal plasma.Design and methods. PSA in seminal plasma from men attending a fertility clinic and healthy controls was analyzed using SDS-PAGE, Western blotting and mass spectrometry. Results: Using mass spectrometry, different forms of PSA could be identified in 1-9 bands seen on SDS-PAGE analysis of the respective sample. However, a majority of these molecular forms of PSA were not observed on Western blots. Enzymatic activity of PSA isoforms was demonstrated by sequencing data in zymogram gels. Multivariate analysis of clinical data revealed well-separated patient groups. Conclusions: We demonstrated that PSA in seminal plasma occurs in several isoforms, yet not all were detectable using an antibody based clinical routine method. The heterogeneity of PSA expression might be of clinical significance, by an improved patient phenotyping. © 2011 Elsevier B.V.
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