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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Direct Synthesis of mTRAQ ® Reagent Labeled PeptidesUsing the 433A Peptide SynthesizerStephan Rawer 1 , Andrea Hartmann 2 , and Matthias Glückmann 31 Life Technologies, Applied Biosystems Deutschland GmbH, D-64293, Darmstadt,Germany; 2 Sandoz GmbH, A-6259, Kundl, Austria; 3 AB SCIEX Germany GmbH,D-64293, Darmstadt, GermanyIntroductionSynthetic peptides are the major tools as quantitative markers for Protein Biomarkerexpression analysis. Relative quantification of low abundant proteins is very important toidentify changes in the pathways of the cell status. In recent years the mTRAQ ® reagentlabeling technique has been established as a powerful method in proteomics. Forquantitation in MS, purified synthetic peptides with mTRAQ ® reagent labels are used asinternal standard. Tryptic digestion leads to peptides with arginine and lysine at theC-terminus. The mTRAQ ® reagent is chemically the N-succinimide ester of 4-Methylpiperazine-1-ylacetic acid, which reacts with the amino groups of the peptides. Possiblereacting candidates are the N-terminus and the -amino group of the lysine. After mTRAQ ®reagent labeling, the peptides with arginine will have one and the peptides with a lysinewill have two labels. The common strategy of labeling synthetic peptides in solutionexhibits problems during labeling due to solubility, structural hindrance and side reactions.These problems will be addressed by direct synthesis of the mTRAQ ® reagent labeledpeptides (Figure 1).Fig. 1. Coupling of 4-Methyl-piperazine-1-yl acetic acid to a peptide with a Lysine at theC-terminus.Results and DiscussionIn contrast to the literature we found in our experiments that the removal of the ivDdegroup needs 8% hydrazine [1-3]. An advantage of this side chain protecting group is thepossibility of monitoring at = 290 nm. The modified synthesis was successfully appliedon a 433A peptide synthesizer with UV-monitoring (Figure 2). Additionally thedeprotection of the ivDde-group could be detected at = 301 nm and optimized by thefeedback mechanism. So there was no additional set up needed during synthesis. Thesynthesis described here can be applied in the same way to iTRAQ ® reagent labelledpeptides.In this proof-of-principle study we have chosen the RNA polymerase multi-proteincomplex from Bacillus subtilis [4]. Because of its close relationship to importantpathogens, e. g. Staphylococcus aureus or Bacillus subtilis it became an importantparadigm for gram positive bacteria. The RNA polymerase multi-protein complex plays acrucial role during transcription of all classes of RNAs in bacteria and shows a highhomology between prokaroytic and eukaryotic organisms.150