Abstract
Mass spectrometry-based proteomic analysis of specimens derived from mammalian cell cultures and tissues provides informative views into the complex molecular mechanisms of cellular processes. Good control, efficiency, and reproducibility of protein extraction from cells and tissues are essential aspects for downstream proteomic assays, as well as for other diverse biological and basic research applications. However, common cell lysis techniques suffer from multiple limitations including the lack of reproducibility in protein extraction, covalent alteration of the sample molecules, and misrepresentation of subpopulations of cellular proteins, among others. In this work we have examined the effect of altering both the lysis buffer and the mechanism of lysis in order to enhance proteomic coverage from a cell culture sample. Ultra-high hydrostatic pressure cycling and the addition of fluorinated alcohol in the lysis buffer have been tested due to their proposed individual abilities to solubilize and change conformations of proteins in order to facilitate downstream enzymatic digestion and LC-MS/MS-based proteomic analysis. The resulting data shows that both changes in the cell lysis method demonstrate enrichment for different classes of proteins and protein classes, including membrane- and organelle-associated proteins, meanwhile increasing the total number of recovered proteins and peptides when compared to the conventional lysis methods.
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References
Annan, R.S., Huddleston, M.J., Verma, R., Deshaies, R.J., and Carr, S.A. (2001). A multidimensional electrospray MS-based approach to phosphopeptide mapping. Anal Chem 73, 393–404.
Blonder, J., Chan, K.C., Issaq, H.J., and Veenstra, T.D. (2006). Identification of membrane proteins from mammalian cell/tissue using methanol-facilitated solubilization and tryptic digestion coupled with 2D-LC-MS/MS. Nat Protoc 1, 2784–2790.
Elias, J.E., and Gygi, S.P. (2007). Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods 4, 207–214.
Freeman, E., and Ivanov, A.R. (2010). Proteomics Under Pressure: Development of Essential Sample Preparation Techniques in Proteomics Using Ultra-High Hydrostatic Pressure (Boston, MA, Harvard School of Public Health).
Gast, K., Siemer, A., Zirwer, D., and Damaschun, G. (2001). Fluoroalcohol-induced structural changes of proteins: Some aspects of cosolvent-protein interactions. Eur Biophys J 30, 273–283.
Griffin, N.M., and Schnitzer, J.E. (2010). Overcoming key technological challenges in using mass spectrometry for mapping cell surfaces in tissues. Mol Cell Proteomics 10(2), R110.000935.
Gross, V., Carlson, G., Kwan, A.T., Smejkal, G., Freeman, E., Ivanov, A.R., and Lazarev, A. (2008). Tissue fractionation by hydrostatic pressure cycling technology: The unified sample preparation technique for systems biology studies. J Biomol Tech 19, 189–199.
Gross, M., and Jaenicke, R. (1994). Proteins under pressure. The influence of high hydrostatic pressure on structure, function and assembly of proteins and protein complexes. Eur J Biochem 221, 617–630.
Kyte, J., and Doolittle, R.F. (1982). A simple method for displaying the hydropathic character of a protein. J Mol Biol 157, 105–132.
Lopez-Ferrer, D., Petritis, K., Hixson, K.K., Heibeck, T.H., Moore, R.J., Belov, M.E., Camp, D.G., 2nd, and Smith, R.D. (2008). Application of pressurized solvents for ultrafast trypsin hydrolysis in proteomics: Proteomics on the fly. J Proteome Res 7, 3276–3281.
Redeby, T., Carr, H., Bjork, M., and Emmer, A. (2006). A screening procedure for the solubilization of chloroplast membrane proteins from the marine green macroalga Ulva lactuca using RP-HPLC-MALDI-MS. Int J Biol Macromol 39, 29–36.
Redeby, T., and Emmer, A. (2005). Membrane protein and peptide sample handling for MS analysis using a structured MALDI target. Anal Bioanal Chem 381, 225–232.
Redeby, T., Roeraade, J., and Emmer, A. (2004). Simple fabrication of a structured matrix-assisted laser desorption/ionization target coating for increased sensitivity in mass spectrometric analysis of membrane proteins. Rapid Commun Mass Spectrom 18, 1161–1166.
Ringham, H., Bell, R.L., Smejkal, G.B., Behnke, J., and Witzmann, F.A. (2007). Application of pressure cycling technology to tissue sample preparation for 2-DE. Electrophoresis 28, 1022–1024.
Santoni, V., Molloy, M., and Rabilloud, T. (2000). Membrane proteins and proteomics: Un amour impossible? Electrophoresis 21, 1054–1070.
Sirangelo, I., Dal Piaz, F., Malmo, C., Casillo, M., Birolo, L., Pucci, P., Marino, G., and Irace, G. (2003). Hexafluoroisopropanol and acid destabilized forms of apomyoglobin exhibit structural differences. Biochemistry 42, 312–319.
Smejkal, G.B., Robinson, M.H., Lawrence, N.P., Tao, F., Saravis, C.A., and Schumacher, R.T. (2006). Increased protein yields from Escherichia coli using pressure-cycling technology. J Biomol Tech 17, 173–175.
Smejkal, G.B., Witzmann, F.A., Ringham, H., Small, D., Chase, S.F., Behnke, J., and Ting, E. (2007). Sample preparation for two-dimensional gel electrophoresis using pressure cycling technology. Anal Biochem 363, 309–311.
Acknowledgements
We would like to acknowledge the experimental and analytical contributions of Yelena Margolin for this project. We are also thankful for fruitful discussions and experimental support provided by Vera Gross and Alexander Lazarev of Pressure BioSciences. We thank the Department of Genetics and Complex Diseases at the Harvard School Public Health for funding support.
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Freeman, E.A., Ivanov, A.R. (2011). Pressure-Assisted Lysis of Mammalian Cell Cultures Prior to Proteomic Analysis. In: Ivanov, A., Lazarev, A. (eds) Sample Preparation in Biological Mass Spectrometry. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-0828-0_5
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DOI: https://doi.org/10.1007/978-94-007-0828-0_5
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