Top-down characterization of a native highly intralinked protein: concurrent cleavages of disulfide and protein backbone bonds.
Top-down analysis of proteins has developed rapidly in recent years. However, its application to disulfide-bonded proteins is still limited. Using native chicken lysozyme as a model, we studied the characteristics of collision-induced dissociation (CID) of disulfide-bonded proteins on an LTQ Orbitrap mass spectrometer with electrospray ionization (ESI) in positive mode. For low-charged protein precursor ions with no or limited mobile protons, product ions generated from CID correspond to the concurrent cleavages of disulfide and protein backbone bonds. Up to three disulfide bonds could be easily cleaved with four possible dissociation pathways for each disulfide bond. That led to modifications of the corresponding cysteine residues through addition or subtraction of a hydrogen atom or sulfhydryl group. The protein backbone cleavages mainly occurred at the amide bonds from C-terminal to aspartic acid residues (e.g., ion series of b(18), b(48), y(10), and y(28)), N-C(alpha) bonds from N-terminal to cysteine residues (e.g., c(5), ion series of c(29) and c(63)), and amide bonds from C-terminal to glutamic acid residues (e.g., ion series of b(35)). The characteristics of the top-down analysis for this highly knotted protein will help to understand the general dissociation pattern of disulfide-bonded proteins, which in turn will help to avoid time-consuming bottom-up procedures for the identification of proteins and their modifications.
Chen
Jianzhong
J
Applied Biotechnology Branch, Air Force Research Laboratory, Dayton, Ohio 45433, USA. jianzhongc@yahoo.com
Shiyanov
Pavel
P
Zhang
Liwen
L
Schlager
John J
JJ
Green-Church
Kari B
KB
D000818:Animals; D002645:Chickens; D004220:Disulfides / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry
D004220:Disulfides; D013438:Sulfhydryl Compounds; D006859:Hydrogen; D009113:Muramidase