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. Two-dimensional gel electrophoresis, 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 reversed-phase liquid chromatography (RPLC)-Fourier-transform ion cyclotron resonance (FTICR)-mass spectrometry (MS) is compared with the combination of RPLC fractionation with capillary isoelectric focusing (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.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 890108
- Report Number(s):
- PNNL-SA-49244; 17797; 400412000; TRN: US200621%%571
- Journal Information:
- Electrophoresis, 27(13):2722-2733, Journal Name: Electrophoresis, 27(13):2722-2733
- Country of Publication:
- United States
- Language:
- English
Similar Records
Making broad proteome protein measurements in 1-5 min using high-speed RPLC separations and high-accuracy mass measurments
Automated 20 kpsi RPLC-MS and MS/MS with chromatographic peak capacities of 1000-1500 and capabilities in proteomics and metabolomics
Related Subjects
AUTOMATION
CHROMATOGRAPHY
DETECTION
DIGESTION
ELECTROPHORESIS
FOCUSING
FRACTIONATION
ION CYCLOTRON-RESONANCE
MODIFICATIONS
PEPTIDES
PROTEINS
SPECTROSCOPY
protein separations
FTICR-MS
proteomics
post-translational modifications
Shewanella oneidensis
Environmental Molecular Sciences Laboratory