U.S. Department of Energy

Pacific Northwest National Laboratory

Targeted Quantification of Mutant SPOP Proteins in Prostate Cancer

Introduction 

Recent studies show that somatic mutation at speckle-type POZ protein (SPOP) gene has been identified frequently (up to 15%) in human prostate cancers and is mutually exclusive with prevalent ETS fusions (approximately 50%). However, the vast majority of the current research on SPOP mutation has been performed using genomic techniques, such as exome-sequencing, and the qualitative and quantitative analyses of mutant SPOP at the protein level are still very limited. In addition, the potential of mutant SPOP protein as a biomarker for diagnosis, prognosis or target therapy of prostate cancer remains largely unknown. To address this issue, targeted proteomics assays using high sensitivity SRM mass spectrometry approaches have been developed for quantifying prostate cancer specific mutant SPOP proteins.

 

Methods 

SRM assays were developed for 11 reported mutations of SPOP protein and its non-mutated region. Four different 293T cell lines, with and without expression of three most frequent SPOP mutations in prostate cancer (Y87N, F102C or F133V), were analyzed to evaluate the validity and performance of these assays. After protein extraction, reduction, alkylation, and digestion, peptide mixtures at concentration of 1 µg/µL with spike-in heavy standards were analyzed using either LC-SRM (1 µg loading) or PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing)-SRM (45 µg loading). All the SRM data were generated on Waters nanoACQUITY UPLC coupled to ThermoScientific TSQ Vantage triple quadrupole mass spectrometer and analyzed by Xcalibur and Skyline software.   

 

Preliminary data 

The presence of multiple lysine residues in the SPOP mutation regions precludes the use of conventional trypsin digestion. Arg-C was selected instead due to its superior performance in generating mutation site(s) containing SPOP peptides that are more suitable for SRM analysis compared other proteases (e.g., Asp-N). Although the generated Arg-C peptides are longer and more hydrophobic than typical tryptic peptides, all the LC-SRM assays showed a linear dynamic range of more than two orders of magnitude. The limit of quantification for the mutation site(s) containing peptides ranges from 10 to 100 fmol/mg of total protein in the cell lysate, allowing for confident detection of all three SPOP mutations (Y87N, F102C or F133V) in corresponding mutation positive cell lines, but not in the mutation negative cell lines. The expression of F133V mutation and wild-type SPOP was at much lower levels comparing to that of F102C and Y87N mutations, which agrees with RT-PCR results. It is unknown if this is related to activity of the SPOP protein. In the PRISM-SRM analysis, the commonly used high pH PRISM fractionation method led to significant loss of the hydrophobic SPOP peptides, and hence low pH PRISM was used instead for optimal pre-fractionation/enrichment of the SPOP peptides. Initial PRISM-SRM results showed that the ability to use much greater sample loading (e.g., 45 µg) provided much reduced background and interference, and significantly improved detection of the SPOP peptides compared to the LC-SRM analysis (1 µg sample loading); full characterization of the PRISM-SRM assays is in progress.

 

Novel aspect 

SRM enables multiplexed, isoform-specific detection of mutant SPOP proteins in cell lysates, providing new direction in prostate cancer biomarker development.

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