Increasing Throughput and Data Quality for Proteomics
With the availability of microbial and mammalian genomes combined with dramatic improvements in bioanalytical methods, high-throughput analysis of transcriptomes and proteomes has become a reality for academic and industrial laboratories alike. New technologies have resulted in the discovery of a multitude of novel cellular pathways and interconnective regulatory mechanisms. For instance, the number of drug targets has grown from approximately 500 to the thousands in a short amount of time. While the information from post-genomics techniques is useful in its own right, it does not necessarily accelerate discovery, this is partly because of data management constraints. Bottlenecks also include quality of sample preparation, identification of low abundance compounds, uninterrupted unattended operations, and successful matches of the results with information available in databases.
In this study, we addressed these issues in order to develop robust methods for the discovery of compounds relevant to a product oriented biotechnology environment. Specifically, we developed pre-fractionation methods, deglycosylation protocols, non-radioactive isotopic labeling methods, and improvements in matrix assisted laser desorption (MALDI) matrix techniques to visualize and identify low abundance proteins from complex samples and applied them to a semi-automatic two-dimensional electrophoresis (2-DE) line followed by MALDI time of flight mass spectrometry (MALDI-TOF/MS). The complex exoproteome of a filamentous fungus, Trichoderma reesei, an important production organism for a number of biomass related applications, served as a model system to develop and fine tune most of the methods.
KeywordsBiomass Sulfide Acetonitrile Iodine Cysteine
Unable to display preview. Download preview PDF.
- Börnsen KO (2000) Influence of salts, buffers, detergents, solvents, and matrices on MALDI-MS protein analysis in complex mixtures. Meth. Mol. Biol. 146, 387–404Google Scholar
- Dai Y, Whittal RM, Li L (1999) Two-Layer Sample Preparation: A method for MALDI-MS analysis of complex peptide and protein mixtures. Anal. Chem. 71, 1087–1091Google Scholar
- Dewey RS, Liesch JM, Williams HR, Sugg EE, Dolan CA, Davies P, Mumford RA, Albers-Schonberg G (1992) Purification and characterization by fast-atom-bombardment mass spectrometry of the polymorphonuclear-leucocyte-elastase-generated A alpha (1–21) fragment of fibrinogen from human blood after incubation with calcium ionophore A23187. Biochem J. 281 (pt 2), 519–24PubMedGoogle Scholar
- Dwek RA, Edge CJ, Harvey, DJ, Wormald MR., Parekh RB (1993) Analysis of glycoprotein-associated oligosaccharides. Annu. Rev. Biochem. 62: 65–100Google Scholar
- Fryksdale BG, Jedrzejewski PT, Wong DL, Gaertner AL, Miller BS (2002) Impact of deglycosylation methods on two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry for proteomic analysis. Electrophoresis 23: 2184–2193PubMedCrossRefGoogle Scholar
- Gobom J, Schuerenberg M, Mueller M, Theiss D, Lehrach H, Nordhoff E (2001) a-cyano4-hydroxycinnamic acid affinity sample preparation. A protocol for MALDI-MS peptide analysis in proteomics. Anal. Chem. 73, 434–438Google Scholar
- Grigorieff N, Ceska TA, Downing KH, Baldwin JM, Henderson R (1996) Electron-crystallographic refinement of the structure of bacteriorhodopsin. J. Mol. Biol. 259, 393–421Google Scholar
- Horvath ZS, Corthals GL, Wrigley CW, Margolis 1 (1994) Multifunctional apparatus for electrokinetic processing of proteins. Electrophoresis 15: 968–971Google Scholar
- Kussmann M. and Roepstorff P (2000) Sample preparation techniques for peptides and proteins analyzed by MALDI-MS. Meth. Mol. Biol. 146, 405–424Google Scholar
- Lappalainen A, Siika-Aho M, Kalkkinen N, Fagerstrom R, Tenkanen M (2000) Endoxylanase II from Trichoderma reesei has several isoforms with different isoelectric points. Biotechnol. Appl. Biochem. 31: 61–68Google Scholar
- Maley F, Trimble RB, Tarentino AL, Plummer TH (1989) Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Anal. Biochem. 180: 195–204Google Scholar
- Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P (1992) In-gel digestion of proteins for internal sequence analysis after one-or two-dimensional gel electrophoresis. Anal. Biochem. 203, 173–179Google Scholar
- Sojar H.T, Bahl, OP (1987) A chemical method for the deglycosylation of proteins. Arch. Biochem. Biophys. 259: 52–57Google Scholar
- Speicher K, Kolbas O, Harper S, Speicher DW (2000) Systematic analysis of peptide recoveries from in-gel digestions for protein identifications in proteome studies. J. Biomol. Tech. 11, 74–86Google Scholar
- Stocklin R, Arrighi JF, Hoang-Van K, Vu L, Cerini F, Gilles N, Genet R, Markussen J, Offord RE, Rose K (2000) Positive and negative labeling of human proinsulin, insulin, and C-peptide with stable isotopes. New tools for in vivo pharmacokinetic and metabolic studies. Meth. Mol. Biol. 146, 293–315Google Scholar