Targeted Quantification of Mutant SPOP Proteins in Prostate CancerHui Wang1, Christopher Barbieri2, Jintang He1, Yuqian Gao1, Chaochao Wu1, Athena Schepmoes1, Thomas Fillmore1, Tujin Shi1, Sung-Suk Chae2, Dennis Huang2, Juan Miguel Mosquera2, Wei-Jun Qian1, Richard D. Smith1, Sudhir Srivastava3, Jacob Kagan3, David Camp1, Karin Rodland1, Mark Rubin2, and Tao Liu1

1Pacific Northwest National Laboratory, Richland, WA; 2Institute of Precision Medicine of Weill Cornell Medical College and New York Presbyterian Hospital, New York, NY; 3National Cancer Institute, Bethesda, MD

Introduction

Overview Methods

This work was funded by NCI Early Detection Research Network (EDRN) Interagency Agreement (No. ACN15006-001). Samples were analyzed using capabilities developed under the NIH Director's New Innovator Award Program DP2OD006668, and NIH grants P41GM103493 and U24-CA-160019, and performed in the Environmental Molecular Sciences Laboratory, a U.S. Department of Energy (DOE) Office of Biological and Environmental Research (OBER) national scientific user facility on the Pacific Northwest National Laboratory (PNNL) campus. PNNL is a multiprogram national laboratory operated by Battelle for the DOE under contract DE-AC05-76RL01830.

1. Shi T, et al. Proc Natl Acad Sci USA 109: 15395-15400 (2012).2. He J. et al. Mol Oncol 8: 1169-1180 (2014).3. He J. et al. J Transl Med 13: 418 (2015).

Conclusions

CONTACT: Hui Wang, Ph.D.Biological Sciences DivisionPacific Northwest National LaboratoryE-mail: hui.wang@pnnl.gov

• Missense mutations in speckle-type POZ protein (SPOP) contribute to prostate cancer development by altering the steady-state levels of key components in the androgen-signaling pathway.

• Selected reaction monitoring (SRM)-based targeted quantification enables multiplexed and isoform-specific detection of mutations at protein level.

• SRM assays have been developed for seven most frequent SPOP mutations in the meprin and TRAF (Tumor necrosis factor receptor-associated factor) homology (MATH) domain and two peptides in the non-mutated region.

• PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing)-SRM1 enables the quantification of low abundance endogenous SPOP mutant peptides in the wild type and mutant cell lines.

• Arg-C is superior in generating SPOP mutant peptides for SRM analysis compared to other proteases (e.g., Asp-N).

• The resulting Arg-C peptides are longer and more hydrophobic than tryptic peptides (and contain multiple Lys); however, all the mutation PRISM-SRM assays showed a tested linear dynamic range of 3 orders of magnitude, and the LOQs range from 0.5 to 1.0 fmol/µg of total protein in the cell lysate.

• PRISM-SRM provides increased sample loading and reduced background/interference, resulting in improved sensitivity in detection of the SPOP mutation peptides.

• Applying PRISM-SRM assays to analyze 293T cells with the expression of WT and three most frequent SPOP mutations in prostate cancer led to confident detection of all three SPOP mutations in the positive cell lines, but not in the negative cell lines.

• SRM assays enabled multiplexed, isoform-specific detection of mutant SPOP proteins (and other protein candidates such as TMPRSS2:ERG fusion proteins2,3), which holds great potential in biomarker verification for prostate cancer.

• Somatic mutation at SPOP gene has been identified frequently (up to 15%) in human prostate cancers and is mutually exclusive with prevalent ETS fusions (approximately 50%).

• The vast majority of the research on SPOP mutation has been performed using genomic techniques, such as whole exome-sequencing.

• However, the qualitative and quantitative analyses of mutant SPOP at the protein level are still very limited.

• The potential of the 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 mutant SPOP proteins in prostate cancer.

www.omics.pnl.govCareer Opportunities: For openings in the Integrative Omics Groupat PNNL please visit http://omics.pnl.gov/careers

• SRM assays were developed for the seven most frequent mutations of SPOP protein and its non-mutated region.

• Purified recombinant SPOP protein (OriGene) was used to aid initial test on digestion and peptide selection using a ThermoScientific Velos Orbitrap instrument; surrogate peptides were synthesized by Thermo Scientific.

• Enzymatic digestion was carried out by using Arg-C instead of trypsin to preserve mutation sites, followed by reduction and alkylation.

• Protein digests were either analyzed directly by LC-SRM or fractionated with target peptide fractions of interest selected and analyzed using PRISM-SRM (see Figure 1).

• Peptide mixtures at concentration of 0.2 µg/µL and 1.0 µg/µL with spike-in heavy standards were analyzed using LC-SRM (1 µg loading) and PRISM-SRM (45 µg loading) analysis, respectively.

• Four different 293T cell lines, with and without expression of the three most frequent SPOP mutations in prostate cancer (Y87N, F102C or F133V), were analyzed to evaluate the validity and performance of these assays.

• SRM data were generated on Waters nanoACQUITY UPLC coupled to ThermoScientific TSQ Vantage triple quadrupole mass spectrometer and analyzed by Xcaliburand Skyline software.

Time (min)

QQQ MS

Q1 Q2 Q3

QQQ MS

Q1 Q2 Q3

SRM Signal

Time (min)

3 µm, C18 column(200 µm i.d. × 50 cm)

1.7 µm, C18 column(75 µm i.d. × 25 cm)

iSelection

96 well plate

Conventional SRM analysis

On-line monitoring heavy peptides

Capillary LC (low pH)

Nano LC (low pH)

#2 #6 #10

Fraction multiplexing

Figure 1. PRISM-SRM workflow.

Acknowledgements

References

Results

Figure 4. PRISM-SRM analysis of SPOP peptides in 293T cells expressing WT (A) and mutant Y87N (B), F102C (C) and F133V (D) SPOP proteins.

Table 1. Quantification of mutant peptides in 293T cells expressing WT and mutant (Y87N, F102C and F133V) SPOP proteins (fmol/µg).

Cell line

VNPKGLDEESKDYLSLY87LLLVSCPKSEVR

AKF102KFSILNAKGEETKAMESQR

FVQGKDWGF133KKFIR

LADELGGLWENSR

SLASAQCPFLGPPR

WT Y87N WT F102C WT F133V WT WTWT 1.29 N.Q. 2.43 6.17 21.36

Y87N 61.39 37.67 43.60 179.67 434.64

F102C 98.4 10.86 56.68 188.44 467.78

F133V 4.09 N.Q. 1.82 16.50 29.11

Figure 3. Response curve of three SPOP mutant peptides.

Figure 2. Comparison of Arg-C and Asp-N digestion of SPOP. Red-font amino acids: mutation sites; red bars: spectral counts.

0.1 1 10 100 1000

0.01

0.1

1

10

100R2=0.9972R2=0.9965

VNPKGLDEESKDYLSLNLLLVSCPKSEVR AKCKFSILNAKGEETKAMESQR FVQGKDWGVKKFIR

Peak

Are

a R

atio

Peptide Concentration (fmol/µL)

R2=0.9969

1 11 21 31 41

M S R V P S P P P P A EM S S G P V A E S WC Y T Q I K V V K F S Y MW T I N N F S F C R E E MG E

51 61 71 81 91

V I K S S T F S S G A N D K L KWC L R V N P K G L D E E S K D Y L S L Y L L L V S C P K S E V R A

101 111 121 131 141

K F K F S I L N A K G E E T K AM E S Q R A Y R F V Q G K D WG F K K F I R R D F L L D E A N G L L

151 161 171 181 191

P D D K L T L F C E V S V V Q D S V N I S G Q N T MNMV K V P E C R L A D E L G G L W E N S R F T

201 211 221 231 241

D C C L C V A G Q E F Q A H K A I L A A R S P V F S AM F E H E M E E S K K N R V E I N D V E P E V

251 261 271 281 291

F K E MM C F I Y T G K A P N L D K MA D D L L A A A D K Y A L E R L K VM C E D A L C S N L S V E

301 311 321 331 341

N A A E I L I L A D L H S A D Q L K T Q A V D F I N Y H A S D V L E T S GWK S M V V S H P H L V A

351 361 371

E A Y R S L A S A Q C P F L G P P R K R L K Q S0 50 100