N-terminal protein characterization by mass spectrometry after cyanogen bromide cleavage using combined microscale liquid- and solid-phase derivatization

Heinz Nika, David H. Hawke, Ruth Hogue Angeletti

Research output: Contribution to journalArticle

5 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)19-30
Number of pages12
JournalJournal of Biomolecular Techniques
Volume25
Issue number1
DOIs
StatePublished - 2014

Fingerprint

Cyanogen Bromide
Mass Spectrometry
Peptides
Proteins
Hydroxylamine
Acylation
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Acetylation
Sulfhydryl Compounds
Disulfides
Hydroxyl Radical
Amines
Chromatography
Gels
Equipment and Supplies

Keywords

  • Covalent chromatography
  • N-terminal peptide
  • Peptide identification
  • Reversed-phase supports

ASJC Scopus subject areas

  • Molecular Biology
  • Medicine(all)

Cite this

N-terminal protein characterization by mass spectrometry after cyanogen bromide cleavage using combined microscale liquid- and solid-phase derivatization. / Nika, Heinz; Hawke, David H.; Angeletti, Ruth Hogue.

In: Journal of Biomolecular Techniques, Vol. 25, No. 1, 2014, p. 19-30.

Research output: Contribution to journalArticle

@article{c19e71dd06d547db9ac44982656ebf8f,
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.",
keywords = "Covalent chromatography, N-terminal peptide, Peptide identification, Reversed-phase supports",
author = "Heinz Nika and Hawke, {David H.} and Angeletti, {Ruth Hogue}",
year = "2014",
doi = "10.7171/jbt.14-2501-003",
language = "English (US)",
volume = "25",
pages = "19--30",
journal = "Journal of Biomolecular Techniques",
issn = "1524-0215",
publisher = "Association of Biomolecular Resource Facilities",
number = "1",

}

TY - JOUR

T1 - N-terminal protein characterization by mass spectrometry after cyanogen bromide cleavage using combined microscale liquid- and solid-phase derivatization

AU - Nika, Heinz

AU - Hawke, David H.

AU - Angeletti, Ruth Hogue

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

KW - Covalent chromatography

KW - N-terminal peptide

KW - Peptide identification

KW - Reversed-phase supports

UR - http://www.scopus.com/inward/record.url?scp=84897424359&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84897424359&partnerID=8YFLogxK

U2 - 10.7171/jbt.14-2501-003

DO - 10.7171/jbt.14-2501-003

M3 - Article

VL - 25

SP - 19

EP - 30

JO - Journal of Biomolecular Techniques

JF - Journal of Biomolecular Techniques

SN - 1524-0215

IS - 1

ER -