We studied sequence-dependent retention properties of synthetic 5'-terminal phosphate absent trinucleotides containing adenine, guanine and thymine through reversed-phase liquid chromatography (RPLC) and QSRR modelling. We investigated the influence of separation conditions, namely mobile phase composition (ion interaction agent content, pH and organic constituent content), on sequence-dependent separation by means of ion-interaction RPLC (II-RPLC) using two types of models: experimental design-artificial neural networks (ED-ANN), and linear regression based on molecular dynamics data. The aim was to determine those properties of the above-mentioned analytes responsible for the retention dependence of the sequence. Our results show that there is a deterministic relation between sequence and II-RPLC retention properties of the studied trinucleotides. Further, we can conclude that the higher the content of ion-interaction agent in the mobile phase, the more prominent these properties are. We also show that if we approximate the polar component of solvation energy in QSRR by the electrostatic work in transferring molecules from vacuum to water, and the non-polar component by the solvent accessible surface area, these parameters best describe the retention properties of trinucleotides. There are some exceptions to this finding, namely sequences 5'-NAN-3', 5'-ANN-3', 5'-TGN-3', 5'-NTA-3'and 5'-NGA-3' (N stands for generic nucleotide). Their role is still unknown, but since linear regression including these specific constellations showed a higher observable variance coverage than the model with only the basic descriptors, we may assume that solvent-analyte interactions are responsible for the exceptional behaviour of 5'-NAN-3' & 5'-ANN-3' trinucleotides and some intramolecular interactions of neighbouring nucleobases for 5'-TGN-3', 5'-NTA-3'and 5'-NGA-3' trinucleotides.
- MeSH
- adenin analogy a deriváty izolace a purifikace MeSH
- chromatografie s reverzní fází MeSH
- guanin analogy a deriváty izolace a purifikace MeSH
- kvantitativní vztahy mezi strukturou a aktivitou MeSH
- neuronové sítě MeSH
- oligonukleotidy izolace a purifikace MeSH
- rozpouštědla MeSH
- simulace molekulární dynamiky MeSH
- statická elektřina MeSH
- thymin analogy a deriváty izolace a purifikace MeSH
- voda MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
Retention characteristics of selected synthetic 5'-terminal phosphate absent penta-nucleotides containing adenine, guanine, and thymine were studied in relation to their sequence by hydrophilic interaction chromatography and ion-interaction reversed-phase liquid chromatography. The organic solvent content, pH, and buffer concentration in mobile phases were evaluated as influential separation conditions. Data demonstrate that both compared chromatographic modes can be used to separate synthetic penta-nucleotides according to their nucleotide composition. Moreover, reversed-phase liquid chromatography allows separation according to their sequence. We have found a simple linear additive model to describe the retention order in both separation modes in regard to their sequence. In hydrophilic interaction chromatography, the retention behavior is controlled primarily by the hydrophilicity of involved nucleotides and minimally by their sequence position. For reversed-phase liquid chromatography, the nucleotide hydrophobicity plays an important role in their retention properties and the influence of their location in sequence on the retention increases toward the center and decreases toward the termini. Our results show that the penta-nucleotide sequence, and thus its spatial arrangement induced by the surrounding environment, is highly related to the retention properties, so it may be hypothetically used to read the sequence from the retention properties acquired under particular separation conditions.
- MeSH
- chromatografie kapalinová metody MeSH
- chromatografie s reverzní fází metody MeSH
- hydrofobní a hydrofilní interakce MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- oligonukleotidy chemická syntéza chemie MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Trypsin (EC 3.4.21.4) is the protease of choice for proteome analysis using mass spectrometry of peptides in sample digests. In this work, trypsin from Streptomyces griseus (SGT) was purified to homogeneity from pronase. The enzyme was evaluated in in-gel digestion of protein standards followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analyses of the digests. We recognized a remarkable cleavage performance of SGT. The number of produced and matching tryptic peptides was higher than in the case of commonly used bovine trypsin (BT) and allowed us to obtain higher identification scores in database searches. Interestingly, SGT was found to also generate nonspecific peptides whose sequencing by MALDI-TOF/TOF tandem mass spectrometry (MS/MS) revealed a partial F-X, Y-X, and W-X cleavage specificity. To suppress autolysis, either arginine or arginine plus lysine residues in SGT were modified by chemical reagents. In consequence, the autolytic pattern of SGT was reduced significantly, but specific activity dropped dramatically. As demonstrated by relative quantification of peptides at different times, SGT is more stable at 37 degrees C than is its bovine counterpart. We conclude that SGT represents a convenient alternative for proteomic applications involving protein digestion. Moreover, parallel digestions of sample aliquots by SGT and BT provide the possibility of combining partially different results (unique matching peptides) to improve protein identification.
- MeSH
- bakteriální proteiny metabolismus MeSH
- financování organizované MeSH
- molekulární sekvence - údaje MeSH
- peptidové fragmenty chemie metabolismus MeSH
- peptidové mapování MeSH
- proteom analýza chemie metabolismus MeSH
- proteomika metody MeSH
- reprodukovatelnost výsledků MeSH
- sekvence aminokyselin MeSH
- sekvenční analýza proteinů MeSH
- sekvenční homologie aminokyselin MeSH
- skot MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- stabilita enzymů MeSH
- Streptomyces griseus enzymologie MeSH
- teplota MeSH
- trypsin metabolismus MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
The study of experimental design conjunction with artificial neural networks for optimisation of isocratic ion-pair reverse phase HPLC separation of neuroprotective peptides is reported. Different types of experimental designs (full-factorial, fractional) were studied as suitable input and output data sources for ANN training and examined on mixtures of humanin derivatives. The independent input variables were: composition of mobile phase, including its pH, and column temperature. In case of a simple mixture of two peptides, the retention time of the most retentive component and resolution were used as the dependent variables (outputs). In case of a complex mixture with unknown number of components, number of peaks, sum of resolutions and retention time of ultimate peak were considered as output variables. Fractional factorial experimental design has been proved to produce sufficient input data for ANN approximation and thus further allowed decreasing the number of experiments necessary for optimisation. After the optimal separation conditions were found, fractions with peptides were collected and their analysis using off-line matrix assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS) was performed.
- MeSH
- chemická frakcionace metody MeSH
- financování organizované MeSH
- intracelulární signální peptidy a proteiny genetika izolace a purifikace MeSH
- neuronové sítě MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- substituce aminokyselin MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- MeSH
- financování organizované MeSH
- Publikační typ
- abstrakty MeSH
