TY - JOUR
T1 - C-terminal protein characterization by mass spectrometry using combined micro scale liquid and solid-phase derivatization
AU - Nika, H.
AU - Nieves, E.
AU - Hawke, David H.
AU - Angeletti, Ruth Hogue
PY - 2013/3
Y1 - 2013/3
N2 - 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 ZipTipC18 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.
AB - 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 ZipTipC18 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.
KW - C-terminal peptide identification
KW - C-terminal peptide structural characterization
KW - Multistep derivatization
KW - Reversed-phase supports
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U2 - 10.7171/jbt.13-2401-003
DO - 10.7171/jbt.13-2401-003
M3 - Article
C2 - 23543807
AN - SCOPUS:84875727387
SN - 1524-0215
VL - 24
SP - 17
EP - 31
JO - Journal of Biomolecular Techniques
JF - Journal of Biomolecular Techniques
IS - 1
ER -