TY - JOUR
T1 - N-terminal protein characterization by mass spectrometry using combined microscale liquid and solid-phase derivatization
AU - Nika, Heinz
AU - Angeletti, Ruth Hogue
AU - Hawke, David H.
N1 - Publisher Copyright:
© 2014 ABRF.
PY - 2014/9/1
Y1 - 2014/9/1
N2 - 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.
AB - 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.
KW - Covalent chromatography
KW - Gel-separated protein
KW - Peptide isolation
KW - Protein digest
KW - Reversed-phase support
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U2 - 10.7171/jbt.14-2503-001
DO - 10.7171/jbt.14-2503-001
M3 - Article
C2 - 25187758
AN - SCOPUS:84906961613
SN - 1524-0215
VL - 25
SP - 77
EP - 86
JO - Journal of Biomolecular Techniques
JF - Journal of Biomolecular Techniques
IS - 3
ER -