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Title: Quantification of mutant SPOP proteins in prostate cancer using mass spectrometry-based targeted proteomics

Abstract

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 where multiple lysine residues, posing significant challenges for bottom-up proteomics analysis of the SPOP mutations. 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. 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 range 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 cellmore » 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 activity of the SPOP protein. In summary, PRISM-SRM enables multiplexed, isoform-specific detection of mutant SPOP proteins in cell lysates, which holds great potential in biomarker development for prostate cancer.« less

Authors:
; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ORCiD logo
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
1406773
Report Number(s):
PNNL-SA-125993
Journal ID: ISSN 1479-5876; 49639; 453040220
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Translational Medicine; Journal Volume: 15; Journal Issue: 1
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; 59 BASIC BIOLOGICAL SCIENCES; Mass Spectometry; targeted proteomics; SPOP M; prostate cancer; Biomarker; PRISM-SRM; Environmental Molecular Sciences Laboratory

Citation Formats

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 A., and Liu, Tao. Quantification of mutant SPOP proteins in prostate cancer using mass spectrometry-based targeted proteomics. United States: N. p., 2017. Web. doi:10.1186/s12967-017-1276-7.
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 A., & Liu, Tao. Quantification of mutant SPOP proteins in prostate cancer using mass spectrometry-based targeted proteomics. United States. doi:10.1186/s12967-017-1276-7.
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 A., and Liu, Tao. 2017. "Quantification of mutant SPOP proteins in prostate cancer using mass spectrometry-based targeted proteomics". United States. doi:10.1186/s12967-017-1276-7.
@article{osti_1406773,
title = {Quantification of mutant SPOP proteins in prostate cancer using mass spectrometry-based targeted proteomics},
author = {Wang, Hui and Barbieri, Christopher E. and He, Jintang and Gao, Yuqian and Shi, Tujin and Wu, Chaochao and Schepmoes, Athena A. and Fillmore, Thomas L. and Chae, Sung-Suk and Huang, Dennis and Mosquera, Juan Miguel and Qian, Wei-Jun and Smith, Richard D. and Srivastava, Sudhir and Kagan, Jacob and Camp, David G. and Rodland, Karin D. and Rubin, Mark A. and Liu, Tao},
abstractNote = {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 where multiple lysine residues, posing significant challenges for bottom-up proteomics analysis of the SPOP mutations. 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. 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 range 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 activity of the SPOP protein. In summary, PRISM-SRM enables multiplexed, isoform-specific detection of mutant SPOP proteins in cell lysates, which holds great potential in biomarker development for prostate cancer.},
doi = {10.1186/s12967-017-1276-7},
journal = {Journal of Translational Medicine},
number = 1,
volume = 15,
place = {United States},
year = 2017,
month = 8
}
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