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New method of peptide cleavage based on Edman degradation.

Bąchor R, Kluczyk A, Stefanowicz P, Szewczuk Z - Mol. Divers. (2013)

Bottom Line: Furthermore, the application of a fixed charge tag in the form of 2-(4-aza-1-azoniabicyclo[2.2.2]octylammonium)acetyl group increases ionization efficiency and reduces the detection limit, allowing ESI-MS/MS sequencing of peptides in the subfemtomolar range.The proposed strategy is compatible with standard conditions during one-bead-one-compound peptide library synthesis.The applicability of the developed strategy in combinatorial chemistry was confirmed using a small training library of [Formula: see text]-chymotrypsin substrates.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Chemistry, University of Wrocław, ul. F. Joliot-Curie 14, 50-383 , Wrocław, Poland.

ABSTRACT
A straightforward cleavage method for N- acylated peptides based on the phenylthiohydantoin (PTH) formation is presented. The procedure could be applied to acid-stable resins, such as TentaGel HL-NH[Formula: see text]. We designed a cleavable linker that consists of a lysine residue with the [Formula: see text]-amino group blocked by Boc, whereas the [Formula: see text]-amino group is used for peptide synthesis. After the peptide assembly is completed, the protecting groups in peptide side chains are removed using trifluoroacetic acid, thus liberating also the [Formula: see text]-amino group of the lysine in the linker. Then the reaction with phenyl isothiocyanate followed by acidolysis causes an efficient peptide release from the resin as a stable PTH derivative. Furthermore, the application of a fixed charge tag in the form of 2-(4-aza-1-azoniabicyclo[2.2.2]octylammonium)acetyl group increases ionization efficiency and reduces the detection limit, allowing ESI-MS/MS sequencing of peptides in the subfemtomolar range. The proposed strategy is compatible with standard conditions during one-bead-one-compound peptide library synthesis. The applicability of the developed strategy in combinatorial chemistry was confirmed using a small training library of [Formula: see text]-chymotrypsin substrates.

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Proposed peptide cleavage method based on Edman degradation. BOCtert-butyloxycarbonyl, Fmoc 9-fluorenylmethoxycarbonyl, Pg protecting group, – amino acid residues, Ac acetyl group, PITC phenyl isothiocyanate. A gray ball represents a single resin bead
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Fig1: Proposed peptide cleavage method based on Edman degradation. BOCtert-butyloxycarbonyl, Fmoc 9-fluorenylmethoxycarbonyl, Pg protecting group, – amino acid residues, Ac acetyl group, PITC phenyl isothiocyanate. A gray ball represents a single resin bead

Mentions: We developed a straightforward method of cleavage of N-acylated peptides from TentaGel HL–NH resin based on the well-known PTH formation reaction commonly used in peptide sequencing by the Edman degradation method. The method utilizes an acid cleavable linker consisting of a lysine residue, where the -amino group is blocked with a Boc group whereas the -amino group is used for peptide synthesis according to Fmoc strategy [21]. The deprotection of side chains with TFA at the same time liberates the -amino group of the lysine residue which in turn reacts with PITC. The final form of the linker consists of a lysine residue with the -amino group blocked by a phenylthiocarbonyl group and an N-acylated peptide assembled on the -amino group (Fig. 1). The subsequent acidolysis leading to Edman degradation results in formation of a stable PTH derivative and efficient peptide release from the resin, allowing identification of the peptide sequence by mass spectrometry, which is especially useful in the analysis of hits in OBOC peptide libraries.Fig. 1


New method of peptide cleavage based on Edman degradation.

Bąchor R, Kluczyk A, Stefanowicz P, Szewczuk Z - Mol. Divers. (2013)

Proposed peptide cleavage method based on Edman degradation. BOCtert-butyloxycarbonyl, Fmoc 9-fluorenylmethoxycarbonyl, Pg protecting group, – amino acid residues, Ac acetyl group, PITC phenyl isothiocyanate. A gray ball represents a single resin bead
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3713267&req=5

Fig1: Proposed peptide cleavage method based on Edman degradation. BOCtert-butyloxycarbonyl, Fmoc 9-fluorenylmethoxycarbonyl, Pg protecting group, – amino acid residues, Ac acetyl group, PITC phenyl isothiocyanate. A gray ball represents a single resin bead
Mentions: We developed a straightforward method of cleavage of N-acylated peptides from TentaGel HL–NH resin based on the well-known PTH formation reaction commonly used in peptide sequencing by the Edman degradation method. The method utilizes an acid cleavable linker consisting of a lysine residue, where the -amino group is blocked with a Boc group whereas the -amino group is used for peptide synthesis according to Fmoc strategy [21]. The deprotection of side chains with TFA at the same time liberates the -amino group of the lysine residue which in turn reacts with PITC. The final form of the linker consists of a lysine residue with the -amino group blocked by a phenylthiocarbonyl group and an N-acylated peptide assembled on the -amino group (Fig. 1). The subsequent acidolysis leading to Edman degradation results in formation of a stable PTH derivative and efficient peptide release from the resin, allowing identification of the peptide sequence by mass spectrometry, which is especially useful in the analysis of hits in OBOC peptide libraries.Fig. 1

Bottom Line: Furthermore, the application of a fixed charge tag in the form of 2-(4-aza-1-azoniabicyclo[2.2.2]octylammonium)acetyl group increases ionization efficiency and reduces the detection limit, allowing ESI-MS/MS sequencing of peptides in the subfemtomolar range.The proposed strategy is compatible with standard conditions during one-bead-one-compound peptide library synthesis.The applicability of the developed strategy in combinatorial chemistry was confirmed using a small training library of [Formula: see text]-chymotrypsin substrates.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Chemistry, University of Wrocław, ul. F. Joliot-Curie 14, 50-383 , Wrocław, Poland.

ABSTRACT
A straightforward cleavage method for N- acylated peptides based on the phenylthiohydantoin (PTH) formation is presented. The procedure could be applied to acid-stable resins, such as TentaGel HL-NH[Formula: see text]. We designed a cleavable linker that consists of a lysine residue with the [Formula: see text]-amino group blocked by Boc, whereas the [Formula: see text]-amino group is used for peptide synthesis. After the peptide assembly is completed, the protecting groups in peptide side chains are removed using trifluoroacetic acid, thus liberating also the [Formula: see text]-amino group of the lysine in the linker. Then the reaction with phenyl isothiocyanate followed by acidolysis causes an efficient peptide release from the resin as a stable PTH derivative. Furthermore, the application of a fixed charge tag in the form of 2-(4-aza-1-azoniabicyclo[2.2.2]octylammonium)acetyl group increases ionization efficiency and reduces the detection limit, allowing ESI-MS/MS sequencing of peptides in the subfemtomolar range. The proposed strategy is compatible with standard conditions during one-bead-one-compound peptide library synthesis. The applicability of the developed strategy in combinatorial chemistry was confirmed using a small training library of [Formula: see text]-chymotrypsin substrates.

Show MeSH
Related in: MedlinePlus