Using size exclusion chromatography-RPLC and RPLC-CIEF as two-dimensional separation strategies for protein profiling.
Bottom-up proteomics (analyzing peptides that result from protein digestion) has demonstrated capability for broad proteome coverage and good throughput. However, due to incomplete sequence coverage, this approach is not ideally suited to the study of modified proteins. The modification complement of a protein can best be elucidated by analyzing the intact protein. 2-DE, typically coupled with the analysis of peptides that result from in-gel digestion, is the most frequently applied protein separation technique in MS-based proteomics. As an alternative, numerous column-based liquid phase techniques, which are generally more amenable to automation, are being investigated. In this work, the combination of size-exclusion chromatography (SEC) fractionation with RPLC-Fourier-transform ion cyclotron resonance (FTICR)-MS is compared with the combination of RPLC fractionation with CIEF-FTICR-MS for the analysis of the Shewanella oneidensis proteome. SEC-RPLC-FTICR-MS allowed the detection of 297 proteins, as opposed to 166 using RPLC-CIEF-FTICR-MS, indicating that approaches based on LC-MS provide better coverage. However, there were significant differences in the sets of proteins detected and both approaches provide a basis for accurately quantifying changes in protein and modified protein abundances.
Simpson
David C
DC
Pacific Northwest National Laboratory, Richland, WA 99352, USA.
Ahn
Seonghee
S
Pasa-Tolic
Ljiljana
L
Bogdanov
Bogdan
B
Mottaz
Heather M
HM
Vilkov
Andrey N
AN
Anderson
Gordon A
GA
Lipton
Mary S
MS
Smith
Richard D
RD
D001426:Bacterial Proteins / Q000032:analysis*; D002853:Chromatography, Liquid / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D007525:Isoelectric Focusing / Q000379:methods*; D010446:Peptide Fragments / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D020592:Shewanella / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared
D001426:Bacterial Proteins; D010446:Peptide Fragments