engleski

Automated sample preparation in proteomics, multidimensional chromatography, biotypization and beyond

Currently used mass spectrometry techniques for protein identification and quantification use database matching based on peptide signals recorded in the positive ion mode. Although widely used, such an approach does not always provide additional biological information beyond the standard output, e.g. the organism itself should be known and preset in the database search, mutations and complex posttranslational modifications should be analyzed in separate and time-consuming experiments and identified proteins have a wide range of statistical evaluation (usually, up to 30 % of identified proteins are very close to the significance level and some of the identified proteins with highly significant score are wrongly annotated). Usage of 15 minutes simple peptide derivatization procedure by 5- formylbenzene-1, 3-disulfonic acid (CAF-/+ reagent) results that positive ion mode peptide fragmentation data can be confirmed by negative ion mode fragmentation data and vice versa. In doing so, validity of the protein sequence coverage method is significantly improved, a whole protein database could be searched without defining the organism (MS/MS biotypization) and mutations and complex posttranslational modifications could be deduced in a single experiment (de novo sequencing). In more flexible and less time-consuming proteomics experimental approach, automated sample preparation followed by 2-D chromatography separation and MALDI- MS/MS-/+ analysis could process up to 16 samples per day.

mass spectromety ; biotypization ; microorganisms

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