title: C-terminal protein characterization by mass spectrometry using combined micro scale liquid and solid-phase derivatization.
abstract: A sample preparation method for protein C-terminal peptide isolation has been developed. In this strategy, protein carboxylate glycinamidation was preceded by carboxyamidomethylation and optional α- and ϵ-amine acetylation in a one-pot reaction, followed by tryptic digestion of the modified protein. The digest was adsorbed on ZipTip(C18) pipette tips for sequential peptide α- and ϵ-amine acetylation and 1-ethyl-(3-dimethylaminopropyl) carbodiimide-mediated carboxylate condensation with ethylenediamine. Amino group-functionalized peptides were scavenged on N-hydroxysuccinimide-activated agarose, 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 exchanged directly on the support, eliminating sample transfer between the reaction steps. By this sequence of solid-phase reactions, the C-terminal peptide could be uniquely recognized in mass spectra of unfractionated digests of moderate complexity. The use of the sample preparation method was demonstrated with low-level amounts of a model protein. The C-terminal peptides were selectively retrieved from the affinity support and proved highly suitable for structural characterization by collisionally induced dissociation. The sample preparation method provides for robustness and simplicity of operation using standard equipment readily available in most biological laboratories and is expected 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, NY 10461, USA.
authors.forename: Heinz
authors.initials: H
authors.lastname: Nika
authors.forename: Edward
authors.initials: E
authors.lastname: Nieves
authors.forename: David H
authors.initials: DH
authors.lastname: Hawke
authors.forename: Ruth Hogue
authors.initials: RH
authors.lastname: Angeletti
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D001920:Bradykinin / Q000737:chemistry* / Q000302:isolation & purification; D005345:Fibrinopeptide B / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D060327:Solid-Phase Synthesis Techniques; D014357:Trypsin / Q000737:chemistry
chemical_list: D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; D005345:Fibrinopeptide B; D014357:Trypsin; D001920:Bradykinin
keywords: C-terminal peptide identification; C-terminal peptide structural characterization; multistep derivatization; reversed-phase supports
references.citation: J Biomol Tech. 2005 Dec;16(4):441-52
references.pmid: 16522867
references.citation: Proteomics. 2008 Feb;8(4):673-85
references.pmid: 18214847
references.citation: Anal Chem. 2007 Jan 15;79(2):673-81
references.pmid: 17222036
references.citation: Rapid Commun Mass Spectrom. 2004;18(20):2388-94
references.pmid: 15386632
references.citation: J Biomol Tech. 2000 Jun;11(2):74-86
references.pmid: 19499040
references.citation: Anal Biochem. 2008 Jun 15;377(2):234-42
references.pmid: 18396144
references.citation: J Biomol Tech. 2002 Dec;13(4):228-37
references.pmid: 19498988
references.citation: J Biomol Tech. 2012 Jul;23(2):51-68
references.pmid: 22951960
references.citation: J Mol Biol. 1998 May 8;278(3):599-608
references.pmid: 9600841
references.citation: J Proteome Res. 2005 May-Jun;4(3):801-8
references.pmid: 15952726
references.citation: Trends Cell Biol. 2002 Mar;12(3):146-50
references.pmid: 11859027
references.citation: Rapid Commun Mass Spectrom. 2004;18(8):863-8
references.pmid: 15095354
references.citation: Proteomics. 2008 Apr;8(8):1539-50
references.pmid: 18340634
references.citation: Anal Chem. 2000 Jul 15;72(14):3374-8
references.pmid: 10939415
references.citation: Anal Chem. 2003 Mar 15;75(6):1300-6
references.pmid: 12659189
references.citation: Nat Methods. 2005 Mar;2(3):193-200
references.pmid: 15782188
references.citation: Rapid Commun Mass Spectrom. 2001;15(16):1416-21
references.pmid: 11507753
references.citation: J Chromatogr A. 1997 Feb 21;762(1-2):97-112
references.pmid: 9098970
references.citation: J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Oct 15;874(1-2):101-10
references.pmid: 18838345
references.citation: Rapid Commun Mass Spectrom. 2009 Mar;23(5):611-8
references.pmid: 19165755
references.citation: Biochim Biophys Acta. 1968 Jun 26;160(2):272-4
references.pmid: 5658133
references.citation: Rapid Commun Mass Spectrom. 2006;20(4):694-702
references.pmid: 16444798
references.citation: Nat Methods. 2010 Jul;7(7):508-11
references.pmid: 20526347