title: N-terminal protein characterization by mass spectrometry using combined microscale liquid and solid-phase derivatization.
abstract: A sample-preparation method for N-terminal peptide isolation from protein proteolytic digests has been developed. Protein thiols and primary amines were protected by carboxyamidomethylation and acetylation, respectively, followed by trypsinization. The digest was bound to ZipTip(C18) pipette tips for reaction of the newly generated N-termini with sulfosuccinimidyl-6-[3'-(2-pyridyldithio)-propionamido] hexanoate. The digest was subsequently exposed to hydroxylamine for reversal of hydroxyl group acylation, followed by reductive release of the pyridine-2-thione moiety from the derivatives. The thiol group-functionalized internal and C-terminal peptides were reversibly captured by covalent chromatography on activated thiol sepharose leaving the N-terminal fragment free in solution. The use of the reversed-phase supports as a reaction bed enabled optimization of the serial modification steps for throughput and completeness of derivatization. The use of the sample-preparation method was demonstrated with low picomole amounts of in-solution- and in-gel-digested protein. The N-terminal peptide was selectively retrieved from the affinity support. The sample-preparation method provides for throughput, robustness, and simplicity of operation using standard equipment available in most biological laboratories and is anticipated to be readily expanded to proteome-wide applications.
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: 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
authors.affiliation: MD Anderson Cancer Center, The University of Texas, Houston, Texas 77030, USA.
authors.forename: David H
authors.initials: DH
authors.lastname: Hawke
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D059748:Proteolysis; D040901:Proteomics; D011725:Pyridines / Q000737:chemistry; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; D011725:Pyridines; C037851:thiol-sepharose; D012685:Sepharose; C029347:2-thiopyridine
keywords: covalent chromatography; gel-separated protein; peptide isolation; protein digest; reversed-phase support