title: C-terminal protein characterization by mass spectrometry: isolation of C-terminal fragments from cyanogen bromide-cleaved protein.
abstract: A sample preparation method for protein C-terminal peptide isolation from cyanogen bromide (CNBr) digests has been developed. In this strategy, the analyte was reduced and carboxyamidomethylated, followed by CNBr cleavage in a one-pot reaction scheme. The digest was then adsorbed on ZipTipC18 pipette tips for conjugation of the homoserine lactone-terminated peptides with 2,2'-dithiobis (ethylamine) dihydrochloride, followed by reductive release of 2-aminoethanethiol from the derivatives. The thiol-functionalized internal and N-terminal peptides were scavenged on activated thiol sepharose, leaving the C-terminal peptide in the flow-through fraction. The use of reversed-phase supports as a venue for peptide derivatization enabled facile optimization of the individual reaction steps for throughput and completeness of reaction. Reagents were replaced directly on the support, allowing the reactions to proceed at minimal sample loss. By this sequence of solid-phase reactions, the C-terminal peptide could be recognized uniquely in mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-level amounts of a whole, intact model protein. The C-terminal fragments were retrieved selectively and efficiently from the affinity support. The use of covalent chromatography for C-terminal peptide purification enabled recovery of the depleted material for further chemical and/or enzymatic manipulation. The sample preparation method provides for robustness and simplicity of operation and is anticipated to be expanded to gel-separated proteins and in a scaled-up format to high-throughput protein profiling in complex biological mixtures.
authors.affiliation: Laboratory for Macromolecular Analysis and Proteomics and Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA; and.
authors.forename: Heinz
authors.initials: H
authors.lastname: Nika
authors.affiliation: MD Anderson Cancer Center, University of Texas, Houston, Texas 77030, USA.
authors.forename: David H
authors.initials: DH
authors.lastname: Hawke
authors.affiliation: Laboratory for Macromolecular Analysis and Proteomics and Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA; and.
authors.forename: Ruth Hogue
authors.initials: RH
authors.lastname: Angeletti
mesh_terms: D003488:Cyanogen Bromide / Q000737:chemistry; D013058:Mass Spectrometry; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D059748:Proteolysis
chemical_list: D010446:Peptide Fragments; D011506:Proteins; D003488:Cyanogen Bromide
keywords: C-terminomics; ZipTipC18 pipette tips; aminolysis; covalent chromatography; peptide; serial solid-phase derivatization