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

Heinz Nika, Ruth Hogue Angeletti, David H. Hawke

Research output: Contribution to journalArticle

1 Citation (Scopus)

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 ZipTipC18 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 proteomewide applications.

Original languageEnglish (US)
Pages (from-to)77-86
Number of pages10
JournalJournal of Biomolecular Techniques
Volume25
Issue number3
DOIs
StatePublished - Sep 1 2014

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Mass Spectrometry
Sulfhydryl Compounds
Peptides
Hydroxylamine
Proteins
Acylation
Acetylation
Hydroxyl Radical
Amines
Chromatography
Gels
Equipment and Supplies
sulfosuccinimidyl 6-(3'-(2-pyridyldithio)propionamido)hexanoate
thiol-sepharose
2-thiopyridine

Keywords

  • Covalent chromatography
  • Gel-separated protein
  • Peptide isolation
  • Protein digest
  • Reversed-phase support

ASJC Scopus subject areas

  • Molecular Biology

Cite this

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

In: Journal of Biomolecular Techniques, Vol. 25, No. 3, 01.09.2014, p. 77-86.

Research output: Contribution to journalArticle

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