Skip to main content
SpringerLink
Log in

Quantitation of Heat-Shock Proteins in Clinical Samples Using Mass Spectrometry

  • Protocol
  • First Online:
Molecular Chaperones

Part of the book series: Methods in Molecular Biology ((MIMB,volume 787))

  • 2018 Accesses

Abstract

Mass spectrometry (MS) is a powerful analytical tool for proteomics research and drug and biomarker discovery. MS enables identification and quantification of known and unknown compounds by revealing their structural and chemical properties. Proper sample preparation for MS-based analysis is a critical step in the proteomics workflow because the quality and reproducibility of sample extraction and preparation for downstream analysis significantly impact the separation and identification capabilities of mass spectrometers. The highly expressed proteins represent potential biomarkers that could aid in diagnosis, therapy, or drug development. Because the proteome is so complex, there is no one standard method for preparing protein samples for MS analysis. Protocols differ depending on the type of sample, source, experiment, and method of analysis. Molecular chaperones play significant roles in almost all biological functions due to their capacity for detecting intracellular denatured/unfolded proteins, initiating refolding or denaturation of such malfolded protein sequences and more recently for their role in the extracellular milieu as chaperokines. In this chapter, we describe the latest techniques for quantitating the expression of molecular chaperones in human clinical samples.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Anderson, N. L., Matheson, A. D., and Steiner, S. (2000) Proteomics: applications in basic and applied biology Curr Opin Biotechnol 11, 408–12.

    Google Scholar 

  2. Adkins, J. N., Varnum, S. M., Auberry, K. J., Moore, R. J., Angell, N. H., Smith, R. D., Springer, D. L., and Pounds, J. G. (2002) Toward a human blood serum proteome: ­analysis by multidimensional separation coupled with mass spectrometry Mol Cell Proteomics 1, 947–55.

    Google Scholar 

  3. Diamandis, E. P. (2004) How are we going to discover new cancer biomarkers? A proteomic approach for bladder cancer Clin Chem 50, 793–5.

    Google Scholar 

  4. Lathrop, J. T., Hayes, T. K., Carrick, K., and Hammond, D. J. (2005) Rarity gives a charm: evaluation of trace proteins in plasma and serum Expert Rev Proteomics 2, 393–406.

    Google Scholar 

  5. Edelbroek, P. M., van der Heijden, J., and Stolk, L. M. (2009) Dried blood spot methods in therapeutic drug monitoring: methods, assays, and pitfalls Ther Drug Monit 31, 327–36.

    Article  PubMed  Google Scholar 

  6. Garcia Boy, R., Henseler, J., Mattern, R., and Skopp, G. (2008) Determination of morphine and 6-acetylmorphine in blood with use of dried blood spots Ther Drug Monit 30, 733–9.

    Article  PubMed  CAS  Google Scholar 

  7. Chace, D. H., Millington, D. S., Terada, N., Kahler, S. G., Roe, C. R., and Hofman, L. F. (1993) Rapid diagnosis of phenylketonuria by quantitative analysis for phenylalanine and tyrosine in neonatal blood spots by tandem mass spectrometry Clin Chem 39, 66–71.

    Google Scholar 

  8. Shigematsu, Y., Hata, I., and Tanaka, Y. (2007) Stable-isotope dilution measurement of isovalerylglycine by tandem mass spectrometry in newborn screening for isovaleric acidemia Clin Chim Acta 386, 82–6.

    Google Scholar 

  9. Jemal, M., Ouyang, Z., and Xia, Y. Q. Systematic LC-MS/MS bioanalytical method development that incorporates plasma phospholipids risk avoidance, usage of incurred sample and well thought-out chromatography Biomed Chromatogr 24, 2–19.

    Google Scholar 

  10. Kintz, P., Cirimele, V., Sachs, H., Jeanneau, T., and Ludes, B. (1999) Testing for anabolic ­steroids in hair from two bodybuilders Forensic Sci Int 101, 209–16.

    Google Scholar 

  11. Deshmukh, N., Hussain, I., Barker, J., Petroczi, A., and Naughton, D. P. (2010) Analysis of anabolic steroids in human hair using LC-MS/MS Steroids 75, 710–14.

    Google Scholar 

  12. Jian, W., Edom, R., Weng, N., Zannikos, P., Zhang, Z., and Wang, H. Validation and application of an LC-MS/MS method for quantitation of three fatty acid ethanolamides as biomarkers for fatty acid hydrolase inhibition in human plasma J Chromatogr B Analyt Technol Biomed Life Sci 878, 1687–99.

    Google Scholar 

  13. Lanckmans, K., Sarre, S., Smolders, I., and Michotte, Y. (2008) Quantitative liquid chromatography/mass spectrometry for the ­analysis of microdialysates Talanta 74, 458–69.

    Google Scholar 

  14. McKinney, M. K., and Cravatt, B. F. (2005) Structure and function of fatty acid amide hydrolase Annu Rev Biochem 74, 411–32.

    Google Scholar 

  15. Hsieh, S. Y., Chen, R. K., Pan, Y. H., and Lee, H. L. (2006) Systematical evaluation of the effects of sample collection procedures on low-molecular-weight serum/plasma proteome profiling Proteomics 6, 3189–98.

    Google Scholar 

  16. Banks, R. E., Stanley, A. J., Cairns, D. A., Barrett, J. H., Clarke, P., Thompson, D., and Selby, P. J. (2005) Influences of blood sample processing on low-molecular-weight proteome identified by surface-enhanced laser desorption/ionization mass spectrometry Clin Chem 51, 1637–49.

    Google Scholar 

  17. West-Nielsen, M., Hogdall, E. V., Marchiori, E., Hogdall, C. K., Schou, C., and Heegaard, N. H. (2005) Sample handling for mass spectrometric proteomic investigations of human sera Anal Chem 77, 5114–23.

    Google Scholar 

  18. Fingerhut, R. (2009) False positive rate in newborn screening for congenital adrenal hyperplasia (CAH)-ether extraction reveals two distinct reasons for elevated 17alpha-hydroxyprogesterone (17-OHP) values Steroids 74, 662–5.

    Article  PubMed  CAS  Google Scholar 

  19. van der Heijden, J., de Beer, Y., Hoogtanders, K., Christiaans, M., de Jong, G. J., Neef, C., and Stolk, L. (2009) Therapeutic drug monitoring of everolimus using the dried blood spot method in combination with liquid chromatography-mass spectrometry J Pharm Biomed Anal 50, 664–70.

    Google Scholar 

  20. Mauri, P., De Palma, A., Pozzi, F., Basilico, F., Riva, A., Morazzoni, P., Bombardelli, E., and Rossoni, G. (2006) LC-MS characterization of terpene lactones in plasma of experimental animals treated with Ginkgo biloba extracts Correlation with pharmacological activity J Pharm Biomed Anal 40, 763–8.

    Google Scholar 

  21. Hua, L., Guangji, W., Hao, L., Minwen, H., Haitang, X., Chenrong, H., Jianguo, S., and Tian, L. (2006) Sensitive and selective liquid chromatography-electrospray ionization mass spectrometry analysis of ginkgolide B in dog plasma J Pharm Biomed Anal 40, 88–94.

    Article  PubMed  Google Scholar 

  22. Zhao, Y., Wang, L., Bao, Y., and Li, C. (2007) A sensitive method for the detection and quantification of ginkgo flavonols from plasma Rapid Commun Mass Spectrom 21, 971–81.

    Google Scholar 

  23. Higashi, T., Nishio, T., Uchida, S., Shimada, K., Fukushi, M., and Maeda, M. (2008) Simultaneous determination of 17alpha-hydroxypregnenolone and 17alpha-hydroxyprogesterone in dried blood spots from low birth weight infants using LC-MS/MS J Pharm Biomed Anal 48, 177–82.

    Article  PubMed  CAS  Google Scholar 

  24. Ding, S., Dudley, E., Chen, L., Plummer, S., Tang, J., Newton, R. P., and Brenton, A. G. (2006) Determination of active components of Ginkgo biloba in human urine by capillary high-performance liquid chromatography/mass spectrometry with on-line column-switching purification Rapid Commun Mass Spectrom 20, 3619–24.

    Google Scholar 

  25. Xie, J., Ding, C., Ge, Q., Zhou, Z., and Zhi, X. (2008) Simultaneous determination of ginkgolides A, B, C and bilobalide in plasma by LC-MS/MS and its application to the pharmacokinetic study of Ginkgo biloba extract in rats J Chromatogr B Analyt Technol Biomed Life Sci 864, 87–94.

    Google Scholar 

  26. Wang, X., Zhao, T., Gao, X., Dan, M., Zhou, M., and Jia, W. (2007) Simultaneous determination of 17 ginsenosides in rat urine by ultra performance liquid chromatography-mass spectrometry with solid-phase extraction Anal Chim Acta 594, 265–73.

    Article  PubMed  CAS  Google Scholar 

  27. Yu, K., Ma, Y., Shao, Q., Qu, H., and Cheng, Y. (2007) Simultaneously determination of five ginsenosides in rabbit plasma using solid-phase extraction and HPLC/MS technique after intravenous administration of ‘SHENMAI’ injection J Pharm Biomed Anal 44, 532–9.

    Google Scholar 

  28. Orvisky, E., Drake, S. K., Martin, B. M., Abdel-Hamid, M., Ressom, H. W., Varghese, R. S., An, Y., Saha, D., Hortin, G. L., Loffredo, C. A., and Goldman, R. (2006) Enrichment of low molecular weight fraction of serum for MS analysis of peptides associated with hepatocellular carcinoma Proteomics 6, 2895–902.

    Google Scholar 

  29. Chertov, O., Biragyn, A., Kwak, L. W., Simpson, J. T., Boronina, T., Hoang, V. M., Prieto, D. A., Conrads, T. P., Veenstra, T. D., and Fisher, R. J. (2004) Organic solvent extraction of proteins and peptides from serum as an effective sample preparation for detection and identification of biomarkers by mass spectrometry Proteomics 4, 1195–203.

    Google Scholar 

  30. Echan, L. A., Tang, H. Y., Ali-Khan, N., Lee, K., and Speicher, D. W. (2005) Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma Proteomics 5, 3292–303.

    Google Scholar 

  31. Huang, L., Harvie, G., Feitelson, J. S., Gramatikoff, K., Herold, D. A., Allen, D. L., Amunngama, R., Hagler, R. A., Pisano, M. R., Zhang, W. W., and Fang, X. (2005) Immunoaffinity separation of plasma proteins by IgY microbeads: meeting the needs of proteomic sample preparation and analysis Proteomics 5, 3314–28.

    Google Scholar 

  32. Kaur, P., Reis, M. D., Couchman, G. R., Forjuoh, S. N., Greene Jr, J. F., and Asea, A. (2010) SERPINE1 links obesity and diabetes: a pilot study J Proteomics Bioinform 3, 191–199.

    Google Scholar 

  33. Brand, J., Haslberger, T., Zolg, W., Pestlin, G., and Palme, S. (2006) Depletion efficiency and recovery of trace markers from a multiparameter immunodepletion column Proteomics 6, 3236–42.

    Google Scholar 

  34. Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis, T., and Mann, M. (1996) Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry Nature 379, 466–9.

    Google Scholar 

  35. Maidment, N. T., Brumbaugh, D. R., Rudolph, V. D., Erdelyi, E., and Evans, C. J. (1989) Microdialysis of extracellular endogenous opioid peptides from rat brain in vivo Neuroscience 33, 549–57.

    Google Scholar 

  36. Li, X., Sun, J., Wang, G., Hao, H., Liang, Y., Zheng, Y., Yan, B., and Sheng, L. (2007) Simultaneous determination of panax notoginsenoside R1, ginsenoside Rg1, Rd, Re and Rb1 in rat plasma by HPLC/ESI/MS: platform for the pharmacokinetic evaluation of total panax notoginsenoside, a typical kind of multiple constituent traditional Chinese medicine Biomed Chromatogr 21, 735–46.

    Google Scholar 

  37. Grova, N., Monteau, F., Le Bizec, B., Feidt, C., Andre, F., and Rychen, G. (2005) Determination of phenanthrene and hydroxyphenanthrenes in various biological matrices at trace levels using gas chromatography-mass spectrometry J Anal Toxicol 29, 175–81.

    Google Scholar 

  38. Stokvis, E., Rosing, H., and Beijnen, J. H. (2005) Stable isotopically labeled internal standards in quantitative bioanalysis using liquid chromatography/mass spectrometry: necessity or not? Rapid Commun Mass Spectrom 19, 401–7.

    Article  PubMed  CAS  Google Scholar 

  39. Chan, E. C., Yap, S. L., Lau, A. J., Leow, P. C., Toh, D. F., and Koh, H. L. (2007) Ultra-performance liquid chromatography/time-of-flight mass spectrometry based metabolomics of raw and steamed Panax notoginseng Rapid Commun Mass Spectrom 21, 519–28.

    Google Scholar 

  40. Dooley, K. C. (2003) Tandem mass spectrometry in the clinical chemistry laboratory Clin Biochem 36, 471–81.

    Google Scholar 

  41. Hoogtanders, K., van der Heijden, J., Christiaans, M., Edelbroek, P., van Hooff, J. P., and Stolk, L. M. (2007) Therapeutic drug monitoring of tacrolimus with the dried blood spot method J Pharm Biomed Anal 44, 658–64.

    Google Scholar 

  42. Taylor, P. J. (2005) Matrix effects: the Achilles heel of quantitative high-performance liquid chromatography-electrospray-tandem mass spectrometry Clin Biochem 38, 328–34.

    Article  PubMed  CAS  Google Scholar 

  43. Rogatsky, E., and Stein, D. (2005) Evaluation of matrix effect and chromatography efficiency: new parameters for validation of method development J Am Soc Mass Spectrom 16, 1757–9.

    Google Scholar 

  44. Matuszewski, B. K., Constanzer, M. L., and Chavez-Eng, C. M. (1998) Matrix effect in quantitative LC/MS/MS analyses of biological fluids: a method for determination of finasteride in human plasma at picogram per milliliter concentrations Anal Chem 70, 882–9.

    Google Scholar 

  45. Bonfiglio, R., King, R. C., Olah, T. V., and Merkle, K. (1999) The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds Rapid Commun Mass Spectrom 13, 1175–1185.

    Google Scholar 

  46. Tong, X. S., Wang, J., Zheng, S., Pivnichny, J. V., Griffin, P. R., Shen, X., Donnelly, M., Vakerich, K., Nunes, C., and Fenyk-Melody, J. (2002) Effect of signal interference from dosing excipients on pharmacokinetic screening of drug candidates by liquid chromatography/mass spectrometry Anal Chem 74, 6305–13.

    Google Scholar 

  47. Gray, M. J., Chang, D., Zhang, Y., Liu, J., and Bensoussan, A. Development of liquid chromatography/mass spectrometry methods for the quantitative analysis of herbal medicine in biological fluids: a review Biomed Chromatogr 24, 91–103.

    Google Scholar 

  48. Kobayashi, N., Kazui, M., and Ikeda, T. (2000) Rapid, real-time sampling of R-84760 in blood by in vivo microdialysis with tandem mass spectrometry J Pharm Biomed Anal 21, 1233–42.

    Article  PubMed  CAS  Google Scholar 

  49. Gao, W., Kishida, T., Kimura, K., Kageyama, M., Sumi, M., Yoshikawa, Y., Shibata, N., and Takada, K. (2002) Sensitive and simultaneous determination of HIV protease inhibitors in rat biological samples by liquid chromatography-mass spectrometry Biomed Chromatogr 16, 267–73.

    Google Scholar 

  50. Igarashi, K., Murabayashi, Y., Hotta, K., Kitamura, Y., Kasuya, F., Shiotani, K., Tingyou, L., Miyazaki, A., Tsuda, Y., Okada, Y., and Fukushima, S. (2004) Application of liquid chromatography-tandem mass spectrometry for the determination of opioidmimetics in the brain dialysates from rats treated with opioidmimetics intraperitoneally J Chromatogr B Analyt Technol Biomed Life Sci 806, 53–7.

    Google Scholar 

  51. Sekar, K. S., and Bramer, S. L. (2003) Extended application of an LC-MS/MS method for the analysis of vesnarinone and its metabolites in human urine and dialysate fluid J Pharm Biomed Anal 33, 711–7.

    Google Scholar 

  52. Pickl, K. E., Magnes, C., Bodenlenz, M., Pieber, T. R., and Sinner, F. M. (2007) Rapid online-SPE-MS/MS method for ketoprofen determination in dermal interstitial fluid samples from rats obtained by microdialysis or open-flow microperfusion J Chromatogr B Analyt Technol Biomed Life Sci 850, 432–9.

    Google Scholar 

  53. Prokai, L., Zharikova, A. D., Janaky, T., and Prokai-Tatrai, K. (2000) Exploratory pharmacokinetics and brain distribution study of a neuropeptide FF antagonist by liquid chromatography/atmospheric pressure ionization tandem mass spectrometry Rapid Commun Mass Spectrom 14, 2412–8.

    Google Scholar 

  54. Pang, L. Q., Liang, Q. L., Wang, Y. M., Ping, L., and Luo, G. A. (2008) Simultaneous determination and quantification of seven major phospholipid classes in human blood using normal-phase liquid chromatography coupled with electrospray mass spectrometry and the application in diabetes nephropathy J Chromatogr B Analyt Technol Biomed Life Sci 869, 118–25.

    Google Scholar 

  55. Xia, Y. Q., and Jemal, M. (2009) Phospholipids in liquid chromatography/mass spectrometry bioanalysis: comparison of three tandem mass spectrometric techniques for monitoring plasma phospholipids, the effect of mobile phase composition on phospholipids elution and the association of phospholipids with matrix effects Rapid Commun Mass Spectrom 23, 2125–38.

    Google Scholar 

  56. Little, J. L., Wempe, M. F., and Buchanan, C. M. (2006) Liquid chromatography-mass spectrometry/mass spectrometry method development for drug metabolism studies: Examining lipid matrix ionization effects in plasma J Chromatogr B Analyt Technol Biomed Life Sci 833, 219–30.

    Google Scholar 

  57. Zhang, Z., and Marshall, A. G. (1998) A universal algorithm for fast and automated charge state deconvolution of electrospray mass-to-charge ratio spectra J Am Soc Mass Spectrom 9, 225–33.

    Google Scholar 

  58. Chong, B. E., Hamler, R. L., Lubman, D. M., Ethier, S. P., Rosenspire, A. J., and Miller, F. R. (2001) Differential screening and mass mapping of proteins from premalignant and cancer cell lines using nonporous reversed-phase HPLC coupled with mass spectrometric analysis Anal Chem 73, 1219–27.

    Google Scholar 

  59. Emmett, M. R., Andren, P. E., and Caprioli, R. M. (1995) Specific molecular mass detection of endogenously released neuropeptides using in vivo microdialysis/mass spectrometry J Neurosci Methods 62, 141–7.

    Google Scholar 

  60. Nyitrai, G., Kekesi, K. A., Emri, Z., Szarics, E., Juhasz, G., and Kardos, J. (2003) GABA(B) receptor antagonist CGP-36742 enhances somatostatin release in the rat hippocampus in vivo and in vitro Eur J Pharmacol 478, 111–9.

    Article  PubMed  CAS  Google Scholar 

  61. Pettersson, A., Amirkhani, A., Arvidsson, B., Markides, K., and Bergquist, J. (2004) A feasibility study of solid supported enhanced microdialysis Anal Chem 76, 1678–82.

    Google Scholar 

  62. Jemal, M., Schuster, A., and Whigan, D. B. (2003) Liquid chromatography/tandem mass spectrometry methods for quantitation of mevalonic acid in human plasma and urine: method validation, demonstration of using a surrogate analyte, and demonstration of unacceptable matrix effect in spite of use of a stable isotope analog internal standard Rapid Commun Mass Spectrom 17, 1723–34.

    Google Scholar 

  63. Liu, B., DeFilippo, A. M., and Li, Z. (2002) Overcoming immune tolerance to cancer by heat shock protein vaccines Mol Cancer Ther 1, 1147–51.

    Google Scholar 

  64. Franzen, B., Linder, S., Alaiya, A. A., Eriksson, E., Uruy, K., Hirano, T., Okuzawa, K., and Auer, G. (1996) Analysis of polypeptide expression in benign and malignant human breast lesions: down-regulation of cytokeratins Br J Cancer 74, 1632–8.

    Google Scholar 

  65. Wang, L., Pan, H., and Smith, D. L. (2002) Hydrogen exchange-mass spectrometry: optimization of digestion conditions Mol Cell Proteomics 1, 132–8.

    Google Scholar 

  66. Cheng, G., Cusanovich, M. A., and Wysocki, V. H. (2006) Properties of the dark and signaling states of photoactive yellow protein probed by solution phase hydrogen/deuterium exchange and mass spectrometry Biochemistry 45, 11744–51.

    Google Scholar 

Download references

Acknowledgments

This work was supported in part by a Research Advancement Award from Scott & White Memorial Hospital and Clinic (P.K.), Institutional support from Scott & White Memorial Hospital and Clinic, Texas A&M Health Science Center College of Medicine, the Central Texas Veterans Health Administration, an Endowment from the Cain Foundation, and the US National Institutes of Health grant RO1CA91889 (A.A.).

Author information

Authors and Affiliations

  1. Division of Investigative Pathology, College of Medicine, Scott & White Memorial Hospital and Clinic, Temple, TX, USA

    Punit Kaur & Alexzander Asea

  2. Texas A&M Health Science Center, Temple, TX, USA

    Punit Kaur & Alexzander Asea

Authors
  1. Punit Kaur
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alexzander Asea
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Alexzander Asea .

Editor information

Editors and Affiliations

  1. Beth Israel Deaconess Medical Center, Department of Radiation Oncology, Harvard Medical School, Brookline Ave 330, Boston, 02215, Massachusetts, USA

    Stuart K. Calderwood

  2. National Cancer Institute, Urologic Oncology Branch, National Institutes of Health, Rockville Pike 9000, Bethesda, 20814, Maryland, USA

    Thomas L. Prince

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Kaur, P., Asea, A. (2011). Quantitation of Heat-Shock Proteins in Clinical Samples Using Mass Spectrometry. In: Calderwood, S., Prince, T. (eds) Molecular Chaperones. Methods in Molecular Biology, vol 787. Humana Press. https://doi.org/10.1007/978-1-61779-295-3_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-295-3_14

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-294-6

  • Online ISBN: 978-1-61779-295-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation