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Fundamentals of proteomics
Harant, K. ; Křenková, Alena
The first part of the course will be an introduction to proteomics with the aim to show current methods and tools used for proteomic data acquisition and evaluation. In the first part, we will discuss in abbreviated form all steps of the experimental procedure from 1) designing the experiment to be suitable for subsequent LC/MS analysis, 2) sample preparation using examples of immunoprecipitation and complex lysate, to 3) measurement options using Data Dependent or Data Independent acquisition. The course will also include a critical evaluation of the quality of the measured data. The evaluation will focus on the possibilities of data processing in freeware software such as MaxQuant and DIAnn. We will also briefly introduce you to commercial programs - Proteome Discoverer, PEAKS, Spectronaut. The second part of the course will include an introduction to statistical processing of quantitative proteomic data in Perseus software. Emphasis will be placed on understanding important statistical concepts (data distribution, t-test, ANOVA, p-value, FDR, etc.) and understanding all necessary steps of statistical analysis such as data filtering, data normalization, statistical testing, etc. There will be model data that participants will process during the course.
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Proteomics for beginners
Hubálek, Martin ; Vrbacký, M. ; Křenková, Alena
Proteomic tutorial for the beginners will summarise and present current methods to analyse and evaluate proteomic samples. The tutorial will be divided into two parts. The first part will focus on data accquisition process and interpretation of spectra. The second part will focus on protein quantification.
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Structure and biocatalytic properties of nitrilases from filamentous fungi
Křenková, Alena ; Martínková, Ludmila (advisor) ; Macek, Tomáš (referee) ; Klempier, Norbert (referee)
Filamentous fungi are alongside bacteria a rich source of nitrilases that can be used for biotechnological purposes. There is a large number of sequences of putative nitrilases in databases, but few of these enzymes have been expressed, purified and characterized. According to the sequences in GenBank database, synthetic genes of putative nitrilases from Aspergillus niger CBS 513.88 and Neurospora crassa OR74A were designed, expressed in Escherichia coli and the nitrilases produced were purified and characterized. The effect of chaperone-assisted nitrilase expression was also examined using a set of commercial chaperone plasmids. The structural properties of nitrilases were examined using various methods, such as homology modelling, sedimentation velocity analysis or electron microscopy. Recombinant nitrilase production was compared to their production by native producers - filamentous fungi. The cultivation of native producers was rather difficult and nitrilase production was very low, therefore, the advantage of using prokaryotic expression system was highlighted. Nitrilases with amino acids close to the catalytic cysteine replaced were prepared in order to study the effects of certain amino acids on the activity, chemoselectivity and enantioselectivity of nitrilases. The catalytic properties of...
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Structure and biocatalytic properties of nitrilases from filamentous fungi
Křenková, Alena ; Martínková, Ludmila (advisor) ; Macek, Tomáš (referee) ; Klempier, Norbert (referee)
Filamentous fungi are alongside bacteria a rich source of nitrilases that can be used for biotechnological purposes. There is a large number of sequences of putative nitrilases in databases, but few of these enzymes have been expressed, purified and characterized. According to the sequences in GenBank database, synthetic genes of putative nitrilases from Aspergillus niger CBS 513.88 and Neurospora crassa OR74A were designed, expressed in Escherichia coli and the nitrilases produced were purified and characterized. The effect of chaperone-assisted nitrilase expression was also examined using a set of commercial chaperone plasmids. The structural properties of nitrilases were examined using various methods, such as homology modelling, sedimentation velocity analysis or electron microscopy. Recombinant nitrilase production was compared to their production by native producers - filamentous fungi. The cultivation of native producers was rather difficult and nitrilase production was very low, therefore, the advantage of using prokaryotic expression system was highlighted. Nitrilases with amino acids close to the catalytic cysteine replaced were prepared in order to study the effects of certain amino acids on the activity, chemoselectivity and enantioselectivity of nitrilases. The catalytic properties of...
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