An Integrated Top-Down and Bottom-Up Strategy for Characterization of 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 T) 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.
Key wordsProtein Peptide Proteomics Mass spectrometry LC-MS Top-down Bottom-up FT-ICR MS FTMS Post-translational modification PTM
Portions of this work were supported by the National Center for Research Resources (RR 018522), the National Institute of Allergy and Infectious Diseases (NIH/DHHS through interagency agreement Y1-AI-4894-01), the National Institute of General Medical Sciences (NIGMS, R01 GM063883), and the U. S. Department of Energy (DOE) Office of Biological and Environmental Research. Work was performed in the Environmental Molecular Science Laboratory, a DOE national scientific user facility located on the campus of Pacific Northwest National Laboratory (PNNL) in Richland, Washington. PNNL is a multi-program national laboratory operated by Battelle for the DOE under Contract DE-AC05-76RLO 1830.
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