Increased proteome coverage by combining PAGE and peptide isoelectric focusing: comparative study of gel-based separation approaches.
The in-depth analysis of complex proteome samples requires fractionation of the sample into subsamples prior to LC-MS/MS in shotgun proteomics experiments. We have established a 3D workflow for shotgun proteomics that relies on protein separation by 1D PAGE, gel fractionation, trypsin digestion, and peptide separation by in-gel IEF, prior to RP-HPLC-MS/MS. Our results show that applying peptide IEF can significantly increase the number of proteins identified from PAGE subfractionation. This method delivers deeper proteome coverage and provides a large degree of flexibility in experimentally approaching highly complex mixtures by still relying on protein separation according to molecular weight in the first dimension.
Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Ilian
I
Atanassov
Henning
H
Urlaub
D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D013058:Mass Spectrometry; D010455:Peptides / Q000302:isolation & purification*; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D014357:Trypsin / Q000378:metabolism
D010455:Peptides; D020543:Proteome; D014357:Trypsin
IPGs; PAGE; Peptide IEF; Shotgun proteomics; Technology