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Multivariate analyses for biomarkers hunting and validation through on-tissue bottom-up or in-source decay in MALDI-MSI: application to prostate cancer

Abstract

The large amount of data generated using matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) poses a challenge for data analysis. In fact, generally about 1.108–1.109 values (m/z, I) are stored after a single MALDI-MSI experiment. This imposes processing techniques using dedicated informatics tools to be used since manual data interpretation is excluded. This work proposes and summarizes an approach that utilizes a multivariable analysis of MSI data. The multivariate analysis, such as principal component analysis–symbolic discriminant analysis, can remove and highlight specific m/z from the spectra in a specific region of interest. This approach facilitates data processing and provides better reproducibility, and thus, broadband acquisition for MALDI-MSI should be considered an effective tool to highlight biomarkers of interest. Additionally, we demonstrate the importance of the hierarchical classification of biomarkers by analyzing studies of clusters obtained either from digested or undigested tissues and using bottom-up and in-source decay strategies for in-tissue protein identification. This provides the possibility for the rapid identification of specific markers from different histological samples and their direct localization in tissues. We present an example from a prostate cancer study using formalin-fixed paraffin-embedded tissue.

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References

  1. 1.

    Caprioli RM, Farmer TB, Gile J (1997) Molecular imaging of biological samples: localization of peptides and proteins using MALDI-TOF MS. Anal Chem 69(23):4751–4760

    Article  CAS  Google Scholar 

  2. 2.

    McLean JA, Ridenour WB, Caprioli RM (2007) Profiling and imaging of tissues by imaging ion mobility-mass spectrometry. J Mass Spectrom 42(8):1099–1105

    Article  CAS  Google Scholar 

  3. 3.

    Sugiura Y, Shimma S, Konishi Y, Yamada MK, Setou M (2008) Imaging mass spectrometry technology and application on ganglioside study; visualization of age-dependent accumulation of C20-ganglioside molecular species in the mouse hippocampus. PLoS ONE 3(9):e3232

    Article  Google Scholar 

  4. 4.

    Landgraf RR, Prieto Conaway MC, Garrett TJ, Stacpoole PW, Yost RA (2009) Imaging of lipids in spinal cord using intermediate pressure matrix-assisted laser desorption-linear ion trap/Orbitrap MS. Anal Chem 81(20):8488–8495

    Article  CAS  Google Scholar 

  5. 5.

    Taban IM, Altelaar AF, van der Burgt YE, McDonnell LA, Heeren RM, Fuchser J, Baykut G (2007) Imaging of peptides in the rat brain using MALDI-FTICR mass spectrometry. J Am Soc Mass Spectrom 18(1):145–151

    Article  CAS  Google Scholar 

  6. 6.

    Sugiura Y, Setou M (2009) Imaging mass spectrometry for visualization of drug and endogenous metabolite distribution: toward in situ pharmacometabolomes. J Neuroimmune Pharmacol

  7. 7.

    Khatib-Shahidi S, Andersson M, Herman JL, Gillespie TA, Caprioli RM (2006) Direct molecular analysis of whole-body animal tissue sections by imaging MALDI mass spectrometry. Anal Chem 78(18):6448–6456

    Article  CAS  Google Scholar 

  8. 8.

    Prideaux B, Staab D, Stoeckli M (2010) Applications of MALDI-MSI to pharmaceutical research. Methods Mol Biol 656:405–413

    Google Scholar 

  9. 9.

    Stoeckli MSD, Schweitzer A (2007) Compound and metabolite distribution measured by MALDI mass spectrometric imaging in whole-body tissue sections. Int J Mass Spectrom 260:195–202

    Article  CAS  Google Scholar 

  10. 10.

    Franck J, Arafah K, Barnes A, Wisztorski M, Salzet M, Fournier I (2009) Improving tissue preparation for matrix-assisted laser desorption ionization mass spectrometry imaging. Part 1: using microspotting. Anal Chem 81(19):8193–8202

    Article  CAS  Google Scholar 

  11. 11.

    Lemaire R, Wisztorski M, Desmons A, Tabet JC, Day R, Salzet M, Fournier I (2006) MALDI-MS direct tissue analysis of proteins: improving signal sensitivity using organic treatments. Anal Chem 78(20):7145–7153

    Article  CAS  Google Scholar 

  12. 12.

    Seeley EH, Caprioli RM (2008) Molecular imaging of proteins in tissues by mass spectrometry. Proc Natl Acad Sci USA 105(47):18126–18131

    Article  CAS  Google Scholar 

  13. 13.

    Jardin-Mathe O, Bonnel D, Franck J, Wisztorski M, Macagno E, Fournier I, Salzet M (2008) MITICS (MALDI imaging team imaging computing system): a new open source mass spectrometry imaging software. J Proteomics

  14. 14.

    Stoeckli M, Farmer TB, Caprioli RM (1999) Automated mass spectrometry imaging with a matrix-assisted laser desorption ionization time-of-flight instrument. J Am Soc Mass Spectrom 10(1):67–71

    Article  CAS  Google Scholar 

  15. 15.

    Chaurand P, Schriver KE, Caprioli RM (2007) Instrument design and characterization for high resolution MALDI-MS imaging of tissue sections. J Mass Spectrom 42(4):476–489

    Article  CAS  Google Scholar 

  16. 16.

    Koestler M, Kirsch D, Hester A, Leisner A, Guenther S, Spengler B (2008) A high-resolution scanning microprobe matrix-assisted laser desorption/ionization ion source for imaging analysis on an ion trap/Fourier transform ion cyclotron resonance mass spectrometer. Rapid Commun Mass Spectrom 22(20):3275–3285

    Article  CAS  Google Scholar 

  17. 17.

    Caprioli RM (2008) Perspectives on imaging mass spectrometry in biology and medicine. Proteomics 8(18):3679–3680

    Article  CAS  Google Scholar 

  18. 18.

    El Ayed M, Bonnel D, Longuespee R, Castelier C, Franck J, Vergara D, Desmons A, Tasiemski A, Kenani A, Vinatier D, Day R, Fournier I, Salzet M (2010) MALDI imaging mass spectrometry in ovarian cancer for tracking, identifying, and validating biomarkers. Med Sci Monit 16(8):BR233-45

    Google Scholar 

  19. 19.

    Franck J, Arafah K, Elayed M, Bonnel D, Vergara D, Jacquet A, Vinatier D, Wisztorski M, Day R, Fournier I, Salzet M (2009) MALDI imaging mass spectrometry: state of the art technology in clinical proteomics. Mol Cell Proteomics 8(9):2023–2033

    Article  CAS  Google Scholar 

  20. 20.

    McDonnell LA, Corthals GL, Willems SM, van Remoortere A, van Zeijl RJ, Deelder AM Peptide and protein imaging mass spectrometry in cancer research. J Proteomics 73(10):1921–1944

  21. 21.

    Wisztorski M, Croix D, Macagno E, Fournier I, Salzet M (2008) Molecular MALDI imaging: an emerging technology for neuroscience studies. Dev Neurobiol 68(6):845–858

    Article  CAS  Google Scholar 

  22. 22.

    Wisztorski M, Lemaire R, Stauber J, Menguelet SA, Croix D, Mathe OJ, Day R, Salzet M, Fournier I (2007) New developments in MALDI imaging for pathology proteomic studies. Curr Pharm Des 13(32):3317–3324

    Article  CAS  Google Scholar 

  23. 23.

    Bakry R, Rainer M, Huck CW, Bonn GK (2011) Protein profiling for cancer biomarker discovery using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and infrared imaging: a review. Anal Chim Acta 690(1):26–34

    Google Scholar 

  24. 24.

    Gustafsson JO, Oehler MK, McColl SR, Hoffmann P (2010) Citric acid antigen retrieval (CAAR) for tryptic peptide imaging directly on archived formalin-fixed paraffin-embedded tissue. J Proteome Res 9(9):4315–4328

    Google Scholar 

  25. 25.

    Drake RR, Cazares LH, Jones EE, Fuller TW, Semmes OJ, Laronga C (2011) Challenges to developing proteomic-based breast cancer diagnostics. OMICS (in press)

  26. 26.

    Seeley EH, Caprioli RM (2011) MALDI imaging mass spectrometry of human tissue: method challenges and clinical perspectives. Trends Biotechnol 29(3):136–143

    Google Scholar 

  27. 27.

    Van de Plas R, Ojeda F, Dewil M, Van Den Bosch L, De Moor B, Waelkens E (2007) Prospective exploration of biochemical tissue composition via imaging mass spectrometry guided by principal component analysis. Pac Symp Biocomput 2007:458–469

  28. 28.

    Deininger SO, Ebert MP, Futterer A, Gerhard M, Rocken C (2008) MALDI imaging combined with hierarchical clustering as a new tool for the interpretation of complex human cancers. J Proteome Res

  29. 29.

    Walch A, Rauser S, Deininger SO, Hofler H (2008) MALDI imaging mass spectrometry for direct tissue analysis: a new frontier for molecular histology. Histochem Cell Biol 130(3):421–434

    Article  CAS  Google Scholar 

  30. 30.

    Trim PJ, Atkinson SJ, Princivalle AP, Marshall PS, West A, Clench MR (2008) Matrix-assisted laser desorption/ionisation mass spectrometry imaging of lipids in rat brain tissue with integrated unsupervised and supervised multivariant statistical analysis. Rapid Commun Mass Spectrom 22(10):1503–1509

    Article  CAS  Google Scholar 

  31. 31.

    Franck J, Ayed ME, Wisztorski M, Salzet M, Fournier I (2010) On tissue protein identification improvement by N-terminal peptide derivatization. Methods Mol Biol 656:323–338

    Google Scholar 

  32. 32.

    Franck J, El Ayed M, Wisztorski M, Salzet M, Fournier I (2009) On-tissue N-terminal peptide derivatizations for enhancing protein identification in MALDI mass spectrometric imaging strategies. Anal Chem 81(20):8305–8317

    Article  CAS  Google Scholar 

  33. 33.

    Debois D, Bertrand V, Quinton L, De Pauw-Gillet MC, De Pauw E (2010) MALDI-in source decay applied to mass spectrometry imaging: a new tool for protein identification. Anal Chem 82(10):4036–4045

    Google Scholar 

  34. 34.

    Seeley EH, Oppenheimer SR, Mi D, Chaurand P, Caprioli RM (2008) Enhancement of protein sensitivity for MALDI imaging mass spectrometry after chemical treatment of tissue sections. J Am Soc Mass Spectrom 19(8):1069–1077

    Article  CAS  Google Scholar 

  35. 35.

    Lemaire R, Desmons A, Tabet JC, Day R, Salzet M, Fournier I (2007) Direct analysis and MALDI imaging of formalin-fixed, paraffin-embedded tissue sections. J Proteome Res 6(4):1295–1305

    Article  CAS  Google Scholar 

  36. 36.

    Groseclose MR, Andersson M, Hardesty WM, Caprioli RM (2007) Identification of proteins directly from tissue: in situ tryptic digestions coupled with imaging mass spectrometry. J Mass Spectrom 42(2):254–262

    Article  CAS  Google Scholar 

  37. 37.

    Stauber J, Lemaire R, Franck J, Bonnel D, Croix D, Day R, Wisztorski M, Fournier I, Salzet M (2008) MALDI imaging of formalin-fixed paraffin-embedded tissues: application to model animals of Parkinson disease for biomarker hunting. J Proteome Res 7(3):969–978

    Article  CAS  Google Scholar 

  38. 38.

    Wisztorski M, Franck J, Salzet M, Fournier I (2010) MALDI direct analysis and imaging of frozen versus FFPE tissues: what strategy for which sample? Methods Mol Biol 656:303–322

    Google Scholar 

  39. 39.

    Reiber DC, Grover TA, Brown RS (1998) Identifying proteins using matrix-assisted laser desorption/ionization in-source fragmentation data combined with database searching. Anal Chem 70(4):673–683

    Article  CAS  Google Scholar 

  40. 40.

    Reiber DC, Brown RS, Weinberger S, Kenny J, Bailey J (1998) Unknown peptide sequencing using matrix-assisted laser desorption/ionization and in-source decay. Anal Chem 70(6):1214–1222

    Article  CAS  Google Scholar 

  41. 41.

    Brown RS, Lennon JJ (1995) Sequence-specific fragmentation of matrix-assisted laser-desorbed protein/peptide ions. Anal Chem 67(21):3990–3999

    Article  CAS  Google Scholar 

  42. 42.

    Hardouin J (2007) Protein sequence information by matrix-assisted laser desorption/ionization in-source decay mass spectrometry. Mass Spectrom Rev 26(5):672–682

    Article  CAS  Google Scholar 

  43. 43.

    Takayama M, Tsugita A (2000) Sequence information of peptides and proteins with in-source decay in matrix assisted laser desorption/ionization-time of flight-mass spectrometry. Electrophoresis 21(9):1670–1677

    Article  CAS  Google Scholar 

  44. 44.

    Takayama M (2001) In-source decay characteristics of peptides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J Am Soc Mass Spectrom 12(4):420–427

    Article  CAS  Google Scholar 

  45. 45.

    Suckau D, Resemann A (2003) T3-sequencing: targeted characterization of the N- and C-termini of undigested proteins by mass spectrometry. Anal Chem 75(21):5817–5824

    Article  CAS  Google Scholar 

  46. 46.

    Resemann A, Wunderlich D, Rothbauer U, Warscheid B, Leonhardt H, Fuchser J, Kuhlmann K, Suckau D Top-down de Novo protein sequencing of a 13.6 kDa camelid single heavy chain antibody by matrix-assisted laser desorption ionization-time-of-flight/time-of-flight mass spectrometry. Anal Chem 82(8):3283–3292

  47. 47.

    Demeure K, Quinton L, Gabelica V, De Pauw E (2007) Rational selection of the optimum MALDI matrix for top-down proteomics by in-source decay. Anal Chem 79(22):8678–8685

    Article  CAS  Google Scholar 

  48. 48.

    Groseclose MR, Massion PP, Chaurand P, Caprioli RM (2008) High-throughput proteomic analysis of formalin-fixed paraffin-embedded tissue microarrays using MALDI imaging mass spectrometry. Proteomics 8(18):3715–3724

    Article  CAS  Google Scholar 

  49. 49.

    Ronci M, Bonanno E, Colantoni A, Pieroni L, Di Ilio C, Spagnoli LG, Federici G, Urbani A (2008) Protein unlocking procedures of formalin-fixed paraffin-embedded tissues: application to MALDI-TOF imaging MS investigations. Proteomics 8(18):3702–3714

    Article  CAS  Google Scholar 

  50. 50.

    Djidja MC, Francese S, Loadman PM, Sutton CW, Scriven P, Claude E, Snel MF, Franck J, Salzet M, Clench MR (2009) Detergent addition to tryptic digests and ion mobility separation prior to MS/MS improves peptide yield and protein identification for in situ proteomic investigation of frozen and formalin-fixed paraffin-embedded adenocarcinoma tissue sections. Proteomics 9(10):2750–2763

    Article  CAS  Google Scholar 

  51. 51.

    Tian Y, Zhang H (2010) Isolation of proteins by heat-induced extraction from formalin-fixed, paraffin-embedded tissue and preparation of tryptic peptides for mass spectrometric analysis. Curr Protoc Mol Biol Chapter 10, Unit 10 26 1–7

  52. 52.

    Xu H, Yang L, Wang W, Shi SR, Liu C, Liu Y, Fang X, Taylor CR, Lee CS, Balgley BM (2008) Antigen retrieval for proteomic characterization of formalin-fixed and paraffin-embedded tissues. J Proteome Res 7(3):1098–1108

    Article  CAS  Google Scholar 

  53. 53.

    Shi SR, Liu C, Perez J, Taylor CR (2005) Protein-embedding technique: a potential approach to standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections. J Histochem Cytochem 53(9):1167–1170

    Article  CAS  Google Scholar 

  54. 54.

    D’Amico F, Skarmoutsou E, Stivala F (2009) State of the art in antigen retrieval for immunohistochemistry. J Immunol Methods 341(1–2):1–18

    Article  Google Scholar 

  55. 55.

    Yamashita S (2007) Heat-induced antigen retrieval: mechanisms and application to histochemistry. Prog Histochem Cytochem 41(3):141–200

    Article  CAS  Google Scholar 

  56. 56.

    Shi SR, Cote RJ, Taylor CR (2001) Antigen retrieval techniques: current perspectives. J Histochem Cytochem 49(8):931–937

    Article  CAS  Google Scholar 

  57. 57.

    Shi SR, Cote RJ, Taylor CR (2001) Antigen retrieval immunohistochemistry and molecular morphology in the year 2001. Appl Immunohistochem Mol Morphol 9(2):107–116

    Article  CAS  Google Scholar 

  58. 58.

    Shi SR, Datar R, Liu C, Wu L, Zhang Z, Cote RJ, Taylor CR (2004) DNA extraction from archival formalin-fixed, paraffin-embedded tissues: heat-induced retrieval in alkaline solution. Histochem Cell Biol 122(3):211–218

    Article  CAS  Google Scholar 

  59. 59.

    Shi SR, Shi Y, Taylor CR (2007) Updates on antigen retrieval techniques for immunohistochemistry. Zhonghua Bing Li Xue Za Zhi 36(1):7–10

    CAS  Google Scholar 

  60. 60.

    Shi SR, Liu C, Young L, Taylor C (2007) Development of an optimal antigen retrieval protocol for immunohistochemistry of retinoblastoma protein (pRB) in formalin fixed, paraffin sections based on comparison of different methods. Biotech Histochem 82(6):301–9

    Article  CAS  Google Scholar 

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Acknowledgments

This research was supported by grants from Centre National de la Recherche Scientifique (CNRS), Ministère de L’Education Nationale, de L’Enseignement Supérieur et de la Recherche, Agence Nationale de la Recherche (ANR PCV to IF), Institut du Cancer (INCA to IF), Région Nord-Pas de Calais (PhD financing to DB and RL), the Canadian Institutes of Health Research (CIHR to RD) and the Ministère du Développement Économique, de l’Innovation et de l’Exportation (MDEIE to RD) du Québec and the Fonds de recherche en santé du Québec (FRSQ to RD). RD is a member of the Centre de Recherche Clinique Étienne-Le Bel (Sherbrooke, QC, Canada).

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Correspondence to Michel Salzet.

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Published in the special issue MALDI Imaging with Guest Editor Olivier Laprévote.

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Bonnel, D., Longuespee, R., Franck, J. et al. Multivariate analyses for biomarkers hunting and validation through on-tissue bottom-up or in-source decay in MALDI-MSI: application to prostate cancer. Anal Bioanal Chem 401, 149–165 (2011). https://doi.org/10.1007/s00216-011-5020-5

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Keywords

  • MALDI mass spectrometry imaging
  • Principal component analysis
  • Symbolic discriminant analysis
  • Hierarchical clustering
  • Bottom-up
  • In-source decay
  • Biomarkers
  • Prostate cancer