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Methods Mol Biol. 2011;694:291-304. doi: 10.1007/978-1-60761-977-2_18.

An integrated top-down and bottom-up strategy for characterization of protein isoforms and modifications.

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  • 1Pacific Northwest National Laboratory, Richland, WA, USA.

Abstract

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.

PMID:
21082441
[PubMed - indexed for MEDLINE]
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