Wang, Hui; Barbieri, Christopher E; He, Jintang; Gao, Yuqian; Shi, Tujin; Wu, Chaochao; Schepmoes, Athena A; Fillmore, Thomas L; Chae, Sung-Suk; Huang, Dennis; Mosquera, Juan Miguel; Qian, Wei-Jun; Smith, Richard D; Srivastava, Sudhir; Kagan, Jacob; Camp, David G; Rodland, Karin D; Rubin, Mark Andrew; Liu, Tao (2017). Quantification of mutant SPOP proteins in prostate cancer using mass spectrometry-based targeted proteomics. Journal of translational medicine, 15(1), p. 175. BioMed Central 10.1186/s12967-017-1276-7
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BACKGROUND
Speckle-type POZ protein (SPOP) is an E3 ubiquitin ligase adaptor protein that functions as a potential tumor suppressor, and SPOP mutations have been identified in ~10% of human prostate cancers. However, it remains unclear if mutant SPOP proteins can be utilized as biomarkers for early detection, diagnosis, prognosis or targeted therapy of prostate cancer. Moreover, the SPOP mutation sites are distributed in a relatively short region with multiple lysine residues, posing significant challenges for bottom-up proteomics analysis of the SPOP mutations.
METHODS
To address this issue, PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometry assays have been developed for quantifying wild-type SPOP protein and 11 prostate cancer-derived SPOP mutations.
RESULTS
Despite inherent limitations due to amino acid sequence constraints, all the PRISM-SRM assays developed using Arg-C digestion showed a linear dynamic range of at least two orders of magnitude, with limits of quantification ranged from 0.1 to 1 fmol/μg of total protein in the cell lysate. Applying these SRM assays to analyze HEK293T cells with and without expression of the three most frequent SPOP mutations in prostate cancer (Y87N, F102C or F133V) led to confident detection of all three SPOP mutations in corresponding positive cell lines but not in the negative cell lines. Expression of the F133V mutation and wild-type SPOP was at much lower levels compared to that of F102C and Y87N mutations; however, at present, it is unknown if this also affects the biological activity of the SPOP protein.
CONCLUSIONS
In summary, PRISM-SRM enables multiplexed, isoform-specific detection of mutant SPOP proteins in cell lysates, providing significant potential in biomarker development for prostate cancer.
Item Type: |
Journal Article (Original Article) |
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Division/Institute: |
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > DBMR Forschung Mu35 > Forschungsgruppe Präzisionsonkologie 04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > DBMR Forschung Mu35 > Forschungsgruppe Präzisionsonkologie |
UniBE Contributor: |
Rubin, Mark Andrew |
Subjects: |
500 Science 500 Science > 570 Life sciences; biology |
ISSN: |
1479-5876 |
Publisher: |
BioMed Central |
Language: |
English |
Submitter: |
Marla Rittiner |
Date Deposited: |
08 Mar 2018 13:08 |
Last Modified: |
05 Dec 2022 15:10 |
Publisher DOI: |
10.1186/s12967-017-1276-7 |
PubMed ID: |
28810879 |
Uncontrolled Keywords: |
Biomarker Mass spectrometry PRISM-SRM Prostate cancer SPOP mutation Targeted proteomics |
BORIS DOI: |
10.7892/boris.110759 |
URI: |
https://boris.unibe.ch/id/eprint/110759 |
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