Abstract
Unified schedule for multiple parallel solid-phase synthesis of so-called “difficult” peptides on polypropylene pins was developed. Increase in the efficiency of 9-fluorenyl(methoxycarbonyl) N-terminal amino-protecting group removal was shown to have a greater influence on the accuracy of the “difficult” peptide synthesis than the use of more efficient amino acid coupling reagents such as aminium salts. Hence the unified schedule for multiple parallel solid-phase synthesis of “difficult” peptides included the procedure for N-terminal amino group deprotection modified by applying a more efficient reagent for the deprotection and the standard procedure of amino acid coupling by carbodiimide method with an additional coupling using aminium salts, if necessary. Amino acid coupling with the help of carbodiimide allows to follow the completeness of the coupling via the bromophenol blue indication, thus providing the accuracy of the synthesis and preventing an overexpenditure of expensive reagents. About 100 biotinylated hepatitis C virus envelope protein fragments, most of which represented “difficult” peptides, were successfully obtained by synthesis on pins with the help of the developed unified schedule.
Highlights
Development of proteomic and interactome research linked to the mass-spectral detection and amino acid analysis of peptide fragments of proteins requires extensive development of multiple solid-phase peptide synthesis in order to prepare huge sets of peptides used as calibration standards and as affinity ligands for interactome analysis and interaction site mapping [1,2,3,4,5]
We studied the influence of the Fmoc removal and Fmoc-amino acid coupling conditions on the accuracy of multiple parallel synthesis of peptides with “difficult” sequences in order to elaborate a unified multiple parallel synthesis schedule that allows the correct synthesis of multiple sets of various peptides on pins
PINSOFT2 analysis of the peptide primary structures showed that some of the listed peptides have amino acid sequences, which are prone to the aggregation during the solid-phase synthesis: amino acid residues with β-methyl groups located in a row, amino acid residues with hydrophobic side chains or bulky sidechain protecting groups located one through one, and a big proportion of Gly residues, that are capable to form inter- and intramolecular hydrogen bonds [23, 24]
Summary
Development of proteomic and interactome research linked to the mass-spectral detection and amino acid analysis of peptide fragments of proteins requires extensive development of multiple solid-phase peptide synthesis in order to prepare huge sets of peptides used as calibration standards and as affinity ligands for interactome analysis and interaction site mapping [1,2,3,4,5]. These peptide sets are expected to contain up to several hundreds of peptides including those with modified side-chain functional groups, since the analysis of a single tissue sample from a single organism may require the preparation of more than a hundred of the so-called characteristic peptides (unique fragments of proteins under study). It necessitates a thorough elaboration of multiple parallel peptide synthesis protocols and development of unified procedures that allow obtaining peptide preparations with maximal contents of target products in the shortest time and with the least material and labor expenses
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
