Approaching the complexity of elastase-digested membrane proteomes using off-gel IEF/nLC-MALDI-MS/MS.
Liquid chromatography, coupled with tandem mass spectrometry, is an established method for the identification of proteins from a complex sample. Despite its wide application, the analysis of whole proteomes still represents a challenge to researchers, because of the complexity and dynamic range of protein concentrations in biological samples. The analysis of such samples can be improved by adding a prefractionation step or a combination of orthogonal separation techniques. Off-gel isoelectric focusing (OGE) has successfully been used for prefractionation of a tryptic digest prior to nLC separation. In contrast to previous published results, we present a complete glycerol-free OGE for the analysis of purple membranes and Corynebacterium glutamicum membranes using the less-specific enzyme elastase. More than 85% of the identified unique peptides were found in solely one fraction, with very little carryover. These results are in accordance with those published for tryptic peptides. Therefore, OGE can be used as an effective prefractionation method in a multidimensional separation experiment of nontryptic membrane peptides.
Arrey
Tabiwang N
TN
Cluster of Excellence, Macromolecular Complexes, Institute of Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
Rietschel
Benjamin
B
Papasotiriou
Dimitrios G
DG
Bornemann
Sandra
S
Baeumlisberger
Dominic
D
Karas
Michael
M
Meyer
Bjoern
B
D001426:Bacterial Proteins / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D048230:Corynebacterium glutamicum / Q000378:metabolism; D007525:Isoelectric Focusing / Q000379:methods*; D008565:Membrane Proteins / Q000378:metabolism*; D010196:Pancreatic Elastase / Q000378:metabolism*; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D020543:Proteome; D010196:Pancreatic Elastase