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Coupling protein complex analysis to peptide based proteomics
- Source :
- Journal of chromatography. A. 1217(49)
- Publication Year :
- 2010
-
Abstract
- Proteolysis is a central component of most proteomics methods. Unfortunately much of the information relating to the structural diversity of proteins is lost during digestion. This paper describes a method in which the native proteome of yeast was subjected to preliminary fractionation by size exclusion chromatography (SEC) prior to trypsin digestion of SEC fractions and reversed phase chromatography-mass spectral analysis to identify tryptic peptides thus generated. Through this approach proteins associated with other proteins in high molecular mass complexes were recognized and identified. A focus of this work was on the identification of Hub proteins that associate with multiple interaction partners. A critical component of this strategy is to choose methods and conditions that maximize retention of native structure during the various stages of analysis prior to proteolysis, especially during cell lysis. Maximum survival of protein complexes during lysis was obtained with the French press and bead-beater methods of cell disruption at approximately pH 8 with 200 mM NaCl in the lysis buffer. Structure retention was favored by higher ionic strength, suggesting that hydrophobic effects are important in maintaining the structure of protein complexes. Recovery of protein complexes declined substantially with storage at any temperature, but storage at -20°C was best when low temperature storage was necessary. Slightly lower recovery was obtained with storage at -80°C while lowest recovery was achieved at 4°C. It was concluded that initial fractionation of native proteins in cell lysates by SEC prior to RPC-MS/MS of tryptic digests can be used to recognize and identify proteins in complexes along with their interaction partners in known protein complexes.
- Subjects :
- Proteomics
Lysis
Saccharomyces cerevisiae Proteins
Protein Conformation
Proteolysis
Size-exclusion chromatography
Sodium Chloride
Cell Fractionation
Biochemistry
Analytical Chemistry
Protein structure
Lysis buffer
Protein Interaction Mapping
medicine
Trypsin
Chromatography
medicine.diagnostic_test
Chemistry
Organic Chemistry
Osmolar Concentration
Alcohol Dehydrogenase
Temperature
General Medicine
Hydrogen-Ion Concentration
Proteome
Chromatography, Gel
Cell fractionation
Peptides
Subjects
Details
- ISSN :
- 18733778
- Volume :
- 1217
- Issue :
- 49
- Database :
- OpenAIRE
- Journal :
- Journal of chromatography. A
- Accession number :
- edsair.doi.dedup.....99e0588526e6e2238cea51135b1cc28f