New method of peptide cleavage based on Edman degradation
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New method of peptide cleavage based on Edman degradation Remigiusz Ba˛chor · Alicja Kluczyk · Piotr Stefanowicz · Zbigniew Szewczuk
Received: 30 January 2013 / Accepted: 12 May 2013 / Published online: 21 May 2013 © The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract A straightforward cleavage method for Nacylated peptides based on the phenylthiohydantoin (PTH) formation is presented. The procedure could be applied to acid-stable resins, such as TentaGel HL–NH2 . We designed a cleavable linker that consists of a lysine residue with the α-amino group blocked by Boc, whereas the ε-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 α-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-1azoniabicyclo[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 α-chymotrypsin substrates. Keywords OBOC · Ionization tag · Quaternary ammonium salts · Peptide cleavage method · Phenylthiohydantoin · Edman degradation
Electronic supplementary material The online version of this article (doi:10.1007/s11030-013-9453-y) contains supplementary material, which is available to authorized users. R. Ba˛chor · A. Kluczyk · P. Stefanowicz · Z. Szewczuk (B) Faculty of Chemistry, University of Wrocław, ul. F. Joliot-Curie 14, 50-383 Wrocław, Poland e-mail: [email protected]
Introduction Combinatorial chemistry is widely used in the fast search for new biologically active compounds [1,2]. Particularly useful are combinatorial one-bead-one-compound (OBOC) peptide libraries, obtained by the split-and-mix technique that allows rapid synthesis of millions of compounds on solid support and screening for their biological activity. The screening of OBOC combinatorial peptide libraries is usually carried out with the deprotected peptide remaining attached to the resin bead. After a hit is identified, the respective bead is isolated and the peptide can be sequenced on-resin by Edman degradation or can be cleaved from the bead for sequencing by mass spectrometry. Routinely, the screening is performed on peptides attached to PEGyleted resin such as TentaGel. This hybrid resin is not sensitive to acids or bases [3,4]; therefore, after deprotection of side-chain groups with trifluoroacetic acid (TFA), the peptide is still attached to the solid support. For that reason, a special cleavage
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