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 the capability for broad proteome coverage and good throughput, but is not ideally suited to the discovery and identification of modified proteins. Top-down proteomics (including subjecting intact protein ions to gas-phase dissociation) allows the study of modified proteins, but coverage, sensitivity and throughput are presently problematic. In this work, we describe the combination of bottom-up with intact protein analyses for the characterization of modified proteins. Fractionation at the intact protein level was employed to reduce complexity and increase measurement dynamic range. Bottom-up measurements were used to identify the subset of proteins that were present in each fraction. These identifications were then used in combination with high-accuracy Fourier-transform ion cyclotron resonance (FTICR)-mass spectrometry (MS) intact protein mass measurements to achieve protein and modified-protein identifications. The relative performance of size exclusion chromatography (SEC) fractionation combined with on-line reversed-phase liquid chromatography (RPLC)-FTICR-MS was compared with RPLC fractionation combined with capillary isoelectric focusing (CIEF)-FTICR-MS. Finally, the relative coverage provided by proteomic analyses based on tryptic peptides and intact proteins is considered.
- Research Organization:
- Pacific Northwest National Laboratory (PNNL), Richland, WA (US)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 983740
- Report Number(s):
- PNNL-SA-46424; 400412000
- Journal Information:
- Electrophoresis, 27(13):2722-2733, Journal Name: Electrophoresis, 27(13):2722-2733 Journal Issue: 13 Vol. 27
- Country of Publication:
- United States
- Language:
- English
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