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Development of a Highly Multiplexed SRM Assay for Biomarker Discovery in Formalin-Fixed Paraffin-Embedded Tissues

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Proteomics for Biomarker Discovery

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1959))

Abstract

The search for novel and clinically relevant biomarkers still represents a major clinical challenge and mass-spectrometry-based technologies are essential tools to help in this process. In this application, we demonstrate how selected reaction monitoring (SRM) can be applied in a highly multiplexed way to analyze formalin-fixed paraffin-embedded (FFPE) tissues. Such an assay can be used to analyze numerous samples for narrowing down a list of potential biomarkers to the most relevant candidates. The use of FFPE tissues is of high relevance in this context as large sample collections linked with valuable clinical information are available in hospitals around the world. Here we describe in detail how we proceeded to develop such an assay for 200 proteins in breast tumor FFPE tissues. We cover the selection of suitable peptides, which are different in FFPE compared to fresh frozen tissues and show how we deliberately biased our assay toward proteins with a high probability of being measurable in human clinical samples.

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References

  1. Szasz AM, Gyorffy B, Marko-Varga G (2017) Cancer heterogeneity determined by functional proteomics. Semin Cell Dev Biol 64:132–142. https://doi.org/10.1016/j.semcdb.2016.08.026

    Article  CAS  PubMed  Google Scholar 

  2. Matboli M, El-Nakeep S, Hossam N et al (2016) Exploring the role of molecular biomarkers as a potential weapon against gastric cancer: a review of the literature. World J Gastroenterol 22(26):5896–5908. https://doi.org/10.3748/wjg.v22.i26.5896

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Barbieri CE, Chinnaiyan AM, Lerner SP et al (2017) The emergence of precision urologic oncology: a collaborative review on biomarker-driven therapeutics. Eur Urol 71(2):237–246. https://doi.org/10.1016/j.eururo.2016.08.024

    Article  PubMed  Google Scholar 

  4. Hinestrosa MC, Dickersin K, Klein P et al (2007) Shaping the future of biomarker research in breast cancer to ensure clinical relevance. Nat Rev Cancer 7(4):309–315. https://doi.org/10.1038/nrc2113

    Article  CAS  PubMed  Google Scholar 

  5. Perez-Gracia JL, Sanmamed MF, Bosch A et al (2017) Strategies to design clinical studies to identify predictive biomarkers in cancer research. Cancer Treat Rev 53:79–97. https://doi.org/10.1016/j.ctrv.2016.12.005

    Article  PubMed  Google Scholar 

  6. Ikeda K, Monden T, Kanoh T et al (1998) Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections. J Histochem Cytochem 46(3):397–403. https://doi.org/10.1177/002215549804600314

    Article  CAS  PubMed  Google Scholar 

  7. Hood BL, Conrads TP, Veenstra TD (2006) Mass spectrometric analysis of formalin-fixed paraffin-embedded tissue: unlocking the proteome within. Proteomics 6(14):4106–4114. https://doi.org/10.1002/pmic.200600016

    Article  CAS  PubMed  Google Scholar 

  8. Vincenti DC, Murray GI (2013) The proteomics of formalin-fixed wax-embedded tissue. Clin Biochem 46(6):546–551. https://doi.org/10.1016/j.clinbiochem.2012.10.002

    Article  CAS  PubMed  Google Scholar 

  9. Giusti L, Lucacchini A (2013) Proteomic studies of formalin-fixed paraffin-embedded tissues. Expert Rev Proteomics 10(2):165–177. https://doi.org/10.1586/epr.13.3

    Article  CAS  PubMed  Google Scholar 

  10. Steiner C, Tille JC, Lamerz J et al (2015) Quantification of HER2 by targeted mass spectrometry in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues. Mol Cell Proteomics 14(10):2786–2799. https://doi.org/10.1074/mcp.O115.049049

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hembrough T, Thyparambil S, Liao WL et al (2013) Application of selected reaction monitoring for multiplex quantification of clinically validated biomarkers in formalin-fixed, paraffin-embedded tumor tissue. J Mol Diagn 15(4):454–465. https://doi.org/10.1016/j.jmoldx.2013.03.002

    Article  CAS  PubMed  Google Scholar 

  12. Catenacci DV, Liao WL, Thyparambil S et al (2014) Absolute quantitation of Met using mass spectrometry for clinical application: assay precision, stability, and correlation with MET gene amplification in FFPE tumor tissue. PLoS One 9(7):e100586. https://doi.org/10.1371/journal.pone.0100586

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Nuciforo P, Thyparambil S, Aura C et al (2016) High HER2 protein levels correlate with increased survival in breast cancer patients treated with anti-HER2 therapy. Mol Oncol 10(1):138–147. https://doi.org/10.1016/j.molonc.2015.09.002

    Article  CAS  PubMed  Google Scholar 

  14. Carr SA, Abbatiello SE, Ackermann BL et al (2014) Targeted peptide measurements in biology and medicine: best practices for mass spectrometry-based assay development using a fit-for-purpose approach. Mol Cell Proteomics 13(3):907–917. https://doi.org/10.1074/mcp.M113.036095

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. MacLean B, Tomazela DM, Shulman N et al (2010) Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26(7):966–968. https://doi.org/10.1093/bioinformatics/btq054

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Perou CM, Sorlie T, Eisen MB et al (2000) Molecular portraits of human breast tumours. Nature 406(6797):747–752. https://doi.org/10.1038/35021093

    Article  CAS  PubMed  Google Scholar 

  17. Liu NQ, Stingl C, Look MP et al (2014) Comparative proteome analysis revealing an 11-protein signature for aggressive triple-negative breast cancer. J Natl Cancer Inst 106(2):djt376. https://doi.org/10.1093/jnci/djt376

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Sorlie T, Perou CM, Tibshirani R et al (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A 98(19):10869–10874. https://doi.org/10.1073/pnas.191367098

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Parker JS, Mullins M, Cheang MC et al (2009) Supervised risk predictor of breast cancer based on intrinsic subtypes. J Clin Oncol 27(8):1160–1167. https://doi.org/10.1200/JCO.2008.18.1370

    Article  PubMed  PubMed Central  Google Scholar 

  20. Smid M, Wang Y, Zhang Y et al (2008) Subtypes of breast cancer show preferential site of relapse. Cancer Res 68(9):3108–3114. https://doi.org/10.1158/0008-5472.CAN-07-5644

    Article  CAS  PubMed  Google Scholar 

  21. Rody A, Karn T, Liedtke C et al (2011) A clinically relevant gene signature in triple negative and basal-like breast cancer. Breast Cancer Res 13(5):R97. https://doi.org/10.1186/bcr3035

    Article  PubMed  PubMed Central  Google Scholar 

  22. O’Toole SA, Beith JM, Millar EK et al (2013) Therapeutic targets in triple negative breast cancer. J Clin Pathol 66(6):530–542. https://doi.org/10.1136/jclinpath-2012-201361

    Article  CAS  PubMed  Google Scholar 

  23. Hubner NC, Ren S, Mann M (2008) Peptide separation with immobilized pI strips is an attractive alternative to in-gel protein digestion for proteome analysis. Proteomics 8(23–24):4862–4872. https://doi.org/10.1002/pmic.200800351

    Article  CAS  PubMed  Google Scholar 

  24. Kuster B, Schirle M, Mallick P et al (2005) Scoring proteomes with proteotypic peptide probes. Nat Rev Mol Cell Biol 6(7):577–583. https://doi.org/10.1038/nrm1683

    Article  CAS  PubMed  Google Scholar 

  25. Scherl A, Shaffer SA, Taylor GK et al (2008) Genome-specific gas-phase fractionation strategy for improved shotgun proteomic profiling of proteotypic peptides. Anal Chem 80(4):1182–1191. https://doi.org/10.1021/ac701680f

    Article  CAS  PubMed  Google Scholar 

  26. Maclean B, Tomazela DM, Abbatiello SE et al (2010) Effect of collision energy optimization on the measurement of peptides by selected reaction monitoring (SRM) mass spectrometry. Anal Chem 82(24):10116–10124. https://doi.org/10.1021/ac102179j

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lange V, Picotti P, Domon B et al (2008) Selected reaction monitoring for quantitative proteomics: a tutorial. Mol Syst Biol 4:222. https://doi.org/10.1038/msb.2008.61

    Article  PubMed  PubMed Central  Google Scholar 

  28. Escher C, Reiter L, MacLean B et al (2012) Using iRT, a normalized retention time for more targeted measurement of peptides. Proteomics 12(8):1111–1121. https://doi.org/10.1002/pmic.201100463

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Gallien S, Peterman S, Kiyonami R et al (2012) Highly multiplexed targeted proteomics using precise control of peptide retention time. Proteomics 12(8):1122–1133. https://doi.org/10.1002/pmic.201100533

    Article  CAS  PubMed  Google Scholar 

  30. Sprung RW Jr, Brock JW, Tanksley JP et al (2009) Equivalence of protein inventories obtained from formalin-fixed paraffin-embedded and frozen tissue in multidimensional liquid chromatography-tandem mass spectrometry shotgun proteomic analysis. Mol Cell Proteomics 8(8):1988–1998. https://doi.org/10.1074/mcp.M800518-MCP200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Palmer-Toy DE, Krastins B, Sarracino DA et al (2005) Efficient method for the proteomic analysis of fixed and embedded tissues. J Proteome Res 4(6):2404–2411. https://doi.org/10.1021/pr050208p

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We are grateful to Tom Dunkley and Arno Friedlein for fruitful discussions about SRM assay development and to Thomas McKee for valuable input on breast cancer biomarkers. Furthermore, we are indebted to Gaby Walker for growing the breast cancer cell lines, to Paola Antinori for her help with the isoelectric focusing, to Alex Scherl for running the Perl script on the peptides, and to Marco Berrera for retrieving the isoelectric point for all peptides.

Author information

Author notes

  1. Carine Steiner

    Present address: Late Stage Analytical Development, Small Molecules Technical Development, Roche Innovation Center Basel, F. Hoffmann-La Roche AG, Basel, Switzerland

  2. Paul Cutler

    Present address: Translational Biomarkers and Bioanalysis, Development Sciences, UCB Pharma, Slough, UK

Authors and Affiliations

  1. Division of Laboratory Medicine, Geneva University Hospitals, Geneva, Switzerland

    Carine Steiner & Pierre Lescuyer

  2. Biomarkers, Bioinformatics and Omics, Pharmaceutical Sciences, Roche Pharma Research & Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La Roche AG, Basel, Switzerland

    Carine Steiner, Paul Cutler & Axel Ducret

  3. Clinical Proteomics and Chemistry Group, Faculty of Medicine, University of Geneva, Geneva, Switzerland

    Pierre Lescuyer

  4. Division of Clinical Pathology, Geneva University Hospitals, Geneva, Switzerland

    Jean-Christophe Tille

Authors
  1. Carine Steiner
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  2. Pierre Lescuyer
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  3. Jean-Christophe Tille
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  4. Paul Cutler
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  5. Axel Ducret
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Corresponding author

Correspondence to Carine Steiner .

Editor information

Editors and Affiliations

  1. Université Grenoble Alpes, CEA, Inserm, BGE U1038, Grenoble, France

    Virginie Brun

  2. Université Grenoble Alpes, CEA, Inserm, BGE U1038, Grenoble, France

    Yohann Couté

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Steiner, C., Lescuyer, P., Tille, JC., Cutler, P., Ducret, A. (2019). Development of a Highly Multiplexed SRM Assay for Biomarker Discovery in Formalin-Fixed Paraffin-Embedded Tissues. In: Brun, V., Couté, Y. (eds) Proteomics for Biomarker Discovery. Methods in Molecular Biology, vol 1959. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9164-8_13

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  • DOI: https://doi.org/10.1007/978-1-4939-9164-8_13

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9163-1

  • Online ISBN: 978-1-4939-9164-8

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