Application of Electrospray Ionization Fourier-transform Mass Spectrometry in Top Down Characterization of Proteins (up to 229 KDa) and Profiling of...
- ISBN-10
- 0542980800
- ISBN-13
- 9780542980800
- Pages
- 272
- Language
- English
- Published
- 2007
- Publisher
- Cornell University, Jan.
- Author
- Xuemei Han
Description
For proteins larger than 50 kDa, unusually stable noncovalent tertiary structures in the gas phase have made difficult the tandem MS techniques inside the analyzer cell. Initial inhibition of tertiary structure formation with immediate nozzle-skimmer dissociation efficiently improved dissociation of ubiquitin ions, providing backbone cleavages at 74 out of 75 interresidue bonds. Applying such "prefolding dissociation" to far larger proteins, together with addition of conformer disrupting ESI solution additives, the unusual intractability of large protein ions can be reduced with electrospray additives, heated vaporization, and separate noncovalent and covalent bond dissociation to provide extensive information on sequence and posttranslational modifications. Such dissociative top down approach cleaved 287 interresidue bonds in the termini of a 144 kDa protein, specified previously unidentified disulfide bonds between 8 of 27 cysteines in a 200 kDa protein, and corrected sequence predictions in two proteins, one with 229 kDa.
