Novel Approach for Quantitative Measurement of Matrix Metalloprotease-1 (MMP1) in Human Breast Cancer Cells Using Mass Spectrometry

DOI: 10.4236/jasmi.2013.31006   PDF   HTML   XML   3,407 Downloads   6,403 Views   Citations

Abstract

Identification and quantification of low abundance growth factors and regulators in complex biological samples still present a challenging task in analytical biochemistry. Immunoassays are often used for such purpose but immunoassays face limitation of both availability and qualities of antibody reagents that are necessary for development of immune assays. With genomics data base available, mass spectrometry (MS) can analyze protein tryptic peptides directly for quantitative determination of proteins. In this study, we report a method for detection of matrix metalloproteinase 1 (MMP1), an important extracellular matrix modulator, in human breast cancer cells by quadrupole time-of-flight (Q-TOF) MS. Absolute quantification of MMP1 was conducted using the selected reaction monitoring (SRM) on a triple quadrupole (Triple-Quad) MS via transitions selected from MMP1 tryptic peptides using non isotope labeled MMP1 protein as a titration standard. In comparison with immune based assay, this MS method showed picogram level sensitivity for quantitative determination of MMP1 intotal cell lysates. Our results demonstrated the feasibility of absolute quantification of low abundance proteins using label-free protein standard by mass spectrometry. Therefore, this method provides not only advantages of high sensitivity but also cost saving in comparison with the commonly used mass spectrometry that currently employs isotype labeled proteins for quantitative analysis.

Share and Cite:

M. Fa, K. Hoch, X. Fan, W. Dubinsky, Z. An and N. Zhang, "Novel Approach for Quantitative Measurement of Matrix Metalloprotease-1 (MMP1) in Human Breast Cancer Cells Using Mass Spectrometry," Journal of Analytical Sciences, Methods and Instrumentation, Vol. 3 No. 1, 2013, pp. 54-61. doi: 10.4236/jasmi.2013.31006.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1] I. V. Chernushevich, A. V. Loboda and B. A. Thomson, “An Introduction to Quadrupole-Time-of-Flight Mass Spectrometry,” Journal of Mass Spectrometry, Vol. 36, No. 8, 2001, pp. 849-865. doi:10.1002/jms.207
[2] V. Lange, P. Picotti, B. Domon and R. Aebersold, “Selected Reaction Monitoring for Quantitative Proteomics: A Tutorial,” Molecular Systems Biology, Vol. 4, No. 222, 2008, pp. 1-14. doi:10.1038/msb.2008.61
[3] L. Anderson and C. L. Hunter, “Quantitative Mass Spectrometric Multiple Reaction Monitoring Assays for Major Plasma Proteins,” Molecular & Cellular Proteomics, Vol. 5, No. 4, 2006, pp. 573-588. doi:10.1074/mcp.M500331-MCP200
[4] P. Mitchell, “Proteomics Retrenches,” Nature Biotechnology, Vol. 28, No. 7, 2010, pp. 665-670. doi:10.1038/nbt0710-665
[5] H. Keshishian, T. Addona, M. Burgess, E. Kuhn and S. A. Carr, “Quantitative, Multiplexed Assays for Low Abundance Proteins in Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution,” Molecular & Cellular Proteomics, Vol. 6, No. 12, 2007, pp. 2212-2229. doi:10.1074/mcp.M700354-MCP200
[6] G. Murphy and H. Nagase, “Progress in Matrix Metalloproteinase Research,” Molecular Aspects of Medicine, Vol. 29, No. 5, 2008, pp. 290-308. doi:10.1016/j.mam.2008.05.002
[7] X. Fan, R. J. Brezski, M. Fa, H. Deng, A. Oberholtzer, A. Gonzalez, W. P. Dubinsky, W. R. Strohl, R. E. Jordan, N. Zhang and Z. An, “A Single Proteolytic Cleavage within the Lower Hinge of Trastuzumab Reduces Immune Effector Function and in Vivo Efficacy,” Breast Cancer Research, Vol. 14, No. 4, 2012, p. R116. doi:10.1186/bcr3240
[8] A. Kohrmann, U. Kammerer, M. Kapp, J. Dietl and J. Anacker, “Expression of Matrix Metalloproteinases (MMPs) in Primary Human Breast Cancer and Breast Cancer Cell Lines: New Findings and Review of the Literature,” BMC Cancer, Vol. 9, No. 188, 2009, pp. 1-20. doi:10.1186/1471-2407-9-188
[9] B. K. Choi, D. M. Hercules, T. Zhang and A. I. Gusev, “Comparison of Quadrupole, Time-of-Flight, and Fourier Transform Mass Analyzers for LC-MS Applications,” Current Trends in Mass Spectrometry, Vol. 18, No. 5S, 2003, pp. 524-531.
[10] A. Prakash, D. M. Tomazela, B. Frewen, B. Maclean, G. Merrihew, S. Peterman and M. J. Maccoss, “Expediting the Development of Targeted SRM Assays: Using Data from Shotgun Proteomics to Automate Method Development,” Journal of Proteome Research, Vol. 8, No. 6, 2009, pp. 2733-2739. doi:10.1021/pr801028b
[11] D. S. Kirkpatrick, S. A. Gerber and S. P. Gygi, “The Absolute Quantification Strategy: A General Procedure for the Quantification of Proteins and Post-Translational Modifications,” Methods, Vol. 35, No. 3, 2005, pp. 265-273. doi:10.1016/j.ymeth.2004.08.018
[12] V. Brun, A. Dupuis, A. Adrait, M. Marcellin, D. Thomas, M. Court, F. Vandenesch and J. Garin, “Isotope-Labeled Protein Standards: Toward Absolute Quantitative Proteomics,” Molecular & Cellular Proteomics, Vol. 6, No. 12, 2007, pp. 2139-2149. doi:10.1074/mcp.M700163-MCP200
[13] R. J. Beynon, M. K. Doherty, J. M. Pratt and S. J. Gaskell, “Multiplexed Absolute Quantification in Proteomics Using Artificial QCAT Proteins of Concatenated Signature Peptides,” Nature Methods, Vol. 2, No. 8, 2005, pp. 587-589. doi:10.1038/nmeth774

  
comments powered by Disqus

Copyright © 2020 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.