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Peptide separation using strong anion exchange stationary phase on Liquid Handling Platform

Multidimensional chromatography followed by MALDI- TOF mass spectrometry was used to identify species, sub-species and characterize proteins from different type of cultivated microorganisms. Automated liquid handling platform is a strong tool for fast and precise chromatographic separation which can provide 6 fractions of 96 samples simultaneously in less than 120 minutes. In-house developed cartridges and methods provided us 1-D, 2-D and even 3-D liquid handling chromatography methods dedicated to identify microorganisms to species and sub-species level. Cartridges are printed on 3-D printer and filled with suitable stationary phase for sample separation while method procedures are developed for tailored chromatographic tryptic peptide separation. Quaternary Methyl Ammonium (QMA) is used as a strong anion exchange stationary phase for fractionation of peptides derived from cell extracts followed by Strong Cation Exchange (SCX). All in all, 2-D liquid separation method suited for optimal peptide separation. Firstly, proteins were isolated from cells and digested with trypsin overnight to break down protein bonds into smaller peptides. Each sample of peptides was separated in 6 fractions using QMA stationary phases where obtained fractions were additionally separated in 6 fractions using SCX stationary phases. Later 36 fractions in total of each sample were analyzed using MALDI-TOF mass spectrometry and the raw data were processed using protein database search engine (ProteinReader, IRB). Different stationary phases can be used for the development of a new multidimensional chromatography methods using liquid handling platform. Commercially available cartridges can be easily replaced and loaded with desirable stationary phase where custom made method can be developed. Not less important, such automated platforms are no longer limited and can be used for various type of microbial samples.

peptide separatin ; chromatography ; MALDI-TOF

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