title: N-terminal protein characterization by mass spectrometry after cyanogen bromide cleavage using combined microscale liquid- and solid-phase derivatization.
abstract: A sample preparation method for protein N-terminal peptide isolation from cyanogen bromide (CNBr) protein digests has been developed. In this strategy, the CNBr cleavage was preceded by protein α- and ε-amine acetylation and carboxyamidomethylation in a one-pot reaction scheme. The peptide mixture was adsorbed on ZipTipC18 pipette tips for reaction of the newly generated N-termini with sulfosuccinimidyl-2-(biotinamido) ethyl-1, 3-dithiopropionate. In the subsequent steps, the peptides were exposed in situ to hydroxylamine for reversal of potential hydroxyl group acylation, followed by reductive release of the disulfide-linked biotinamido moiety from the derivatives. The selectively thiol group-functionalized internal and C-terminal peptides were reversibly captured by covalent chromatography on activated thiol-sepharose, leaving the N-terminal fragment in the flow-through fraction. The use of the reversed-phase support as a venue for postcleavage serial modification proved instrumental to ensure throughput and completeness of derivatization. By this sequence of solid-phase reactions, the N-terminal peptide could be recognized uniquely in the MALDI-mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-picomole amounts of model protein. The N-terminal CNBr fragments were retrieved selectively from the affinity support. The sample preparation method provides for robustness and simplicity of operation using standard equipment available in most biological laboratories and is anticipated to be readily expanded to gel-separated proteins.
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: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000737:chemistry*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D059748:Proteolysis; D012685:Sepharose / Q000031:analogs & derivatives; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; C037851:thiol-sepharose; D012685:Sepharose; D003488:Cyanogen Bromide
keywords: N-terminal peptide; covalent chromatography; peptide identification; reversed-phase supports