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Derivatisation of linaclotide and bovine serum albumin with acrylamide and 4-vinylpiridine analyzded by MALDI-TOF/TOF and nanoUPLC coupled to the ESI-QTOF

Proteomics identification and quantification techniques often involve the process of protein derivatization. Binding of different ions or molecules to specific sites of the protein is used to modify structural, ionic, hydrophobic-hydrophilic, and other protein properties. Changes in structural properties allow better chromatographic or ionic separation of protein or protein fragments. At the end, derivatization is carried out to increase overall sensitivity of the analysis. To improve and accelerate protein derivatization procedures of cysteine branches of peptides or proteins, two derivatization reagents, acrylamide and 4- vinylpyridine, were applied [1]. Reactions were optimized using Linaclotide peptide that contains six cysteines interconnected with three disulfide bridges (Figure 1. A). Prior to mass spectrometric analysis derivatization procedure was conducted on bovine serum albumin protein followed by qualitative and quantitative analysis of derivatized tryptic fragments (Figure 1. B). Selective alkylation of the cysteine side branches was achieved after reduction of disulfide bridges. In order to speed up alkylation reaction microwave irradiation was utilized for 8 minutes with radiation power of 180 W. The analysis of peptide reaction mixtures was performed using MALDI-TOF/TOF and nanoUPLC-ESI- QTOF mass spectrometers. Collected spectra were processed with the software search package ProteinLynx Global SERVERTM (PLGS).

Protein derivatization ; Selective alkylation ; MALDI-TOF/TOF ; nanoUPLC-ESI-QTOF ; Proteomics identification

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