Electron Detachment Dissociation for Top-Down Mass Spectrometry of Acidic Proteins

Electron detachment dissociation (EDD) is an emerging mass spectrometry (MS) technique for the primary structure analysis of peptides, carbohydrates, and oligonucleotides. Herein, we explore the potential of EDD for sequencing of proteins of up to 147 amino acid residues by using top-down MS. Sequence coverage ranged from 72 % for Melittin, which lacks carboxylic acid functionalities, to 19 % for an acidic 147-residue protein, to 12 % for Ferredoxin, which showed unusual backbone fragmentation next to cysteine residues. A limiting factor for protein sequencing by EDD is the facile loss of small molecules from amino acid side chains, in particular CO2. Based on the types of fragments observed and fragmentation patterns found, we propose detailed mechanisms for protein backbone cleavage and side chain dissociation in EDD. The insights from this study should further the development of EDD for top-down MS of acidic proteins

Primary Structure of the chicken c−mil protein: identification of domains shared with or absent from the retroviral v−mil protein

The complete primary structure of the protein product of the proto−oncogene c−mil was deduced from the nucleotide sequence of chicken c−mil cDNA clones. The c−mil protein contains 647 amino acid residues and has a calculated molecular weight of 73,132. Based on sequence comparisons with proteins of known or presumed biochemical function, two domains were recognized on the c−mil protein. In the carboxyl−terminal half of the protein, a 250−amino acid segment displays significant homology to the protein kinase domains of the src oncogene protein or of protein kinase C. In the amino−terminal half, a cysteine−rich segment (Cys−X2−Cys−X9−Cys−X2−Cys−X7−Cys−X7−Cys) of the c−mil protein shares significant homology with two similar repetitive domains of protein kinase C. Of the two structural and presumably functional domains of the c−mil protein, only the kinase domain is contained within the carboxyl−terminal 379−amino acid polypeptide encoded by the transduced v−mil allele of avian oncogenic retrovirus MH2. Hence, truncation of the 5' coding region in the course of the transduction and the resulting lack of the authentic amino−terminal domain in the protein product of the transduced allele may be a critical event in changing mil function from physiologic to oncogeni