An integrated top-down and bottom-up strategy for characterization protein isoforms and modifications
Bottom-up and top-down strategies are two commonly used methods for mass spectrometry (MS) based protein identification; each method has its own advantages and disadvantages. In this chapter, we describe an integrated top-down and bottom-up approach facilitated by concurrent liquid chromatography-mass spectrometry (LC-MS) analysis and fraction collection for comprehensive high-throughput intact protein profiling. The approach employs a high resolution reversed phase (RP) LC separation coupled with LC eluent fraction collection and concurrent on-line MS with a high field (12 Tesla) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer. Protein elusion profiles and tentative modified protein identification are made using detected intact protein mass in conjunction with bottom-up protein identifications from the enzymatic digestion and analysis of corresponding LC fractions. Specific proteins of biological interest are incorporated into a target ion list for subsequent off-line gas-phase fragmentation that uses an aliquot of the original collected LC fraction, an aliquot of which was also used for bottom-up analysis.
- 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:
- 1013294
- Report Number(s):
- PNNL-SA-68814; 34504; KP1704020; TRN: US201110%%573
- Resource Relation:
- Related Information: Bioinformatics for Comparative Proteomics: Methods in Molecular Biology, 694:291-304
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
60 APPLIED LIFE SCIENCES
DIGESTION
FRAGMENTATION
ION CYCLOTRON-RESONANCE
MASS SPECTROMETERS
MASS SPECTROSCOPY
MODIFICATIONS
PROTEINS
RESOLUTION
SPECTROSCOPY
TARGETS
protein
peptide
proteomics
mass spectrometry
LC-MS
top-down
bottom-up
FT-ICR MS
FTMS
post-translational modification
PTM
Environmental Molecular Sciences Laboratory