Triple resonance ¹5Ν NMR relaxation experiments for studies of intrinsically disordered proteins.

Description of protein dynamics is known to be essential in understanding their function. Studies based on a well established [Formula: see text] NMR relaxation methodology have been applied to a large number of systems. However, the low dispersion of [Formula: see text] chemical shifts very often observed within intrinsically disordered proteins complicates utilization of standard 2D HN correlated spectra because a limited number of amino acids can be characterized. Here we present a suite of triple resonance HNCO-type NMR experiments for measurements of five [Formula: see text] relaxation parameters ([Formula: see text], [Formula: see text], NOE, cross-correlated relaxation rates [Formula: see text] and [Formula: see text]) in doubly [Formula: see text],[Formula: see text]-labeled proteins. We show that the third spectral dimension combined with non-uniform sampling provides relaxation rates for almost all residues of a protein with extremely poor chemical shift dispersion, the C terminal domain of [Formula: see text]-subunit of RNA polymerase from Bacillus subtilis. Comparison with data obtained using a sample labeled by [Formula: see text] only showed that the presence of [Formula: see text] has a negligible effect on [Formula: see text], [Formula: see text], and on the cross-relaxation rate (calculated from NOE and [Formula: see text]), and that these relaxation rates can be used to calculate accurate spectral density values. Partially [Formula: see text]-labeled sample was used to test if the observed increase of [Formula: see text] [Formula: see text] in the presence of [Formula: see text] corresponds to the [Formula: see text] dipole-dipole interactions in the [Formula: see text],[Formula: see text]-labeled sample.

2D-HPLC and MALDI-TOF/TOF analysis of barley proteins glycated during brewing.

The barley proteins have been the subject of interests of many research groups dealing with barley grains, malt and beer. The proteins which remain intact after harsh malting conditions influence the quality and flavor of beer. The characteristic feature of the proteins present in malt and beer is their extensive modification with carbohydrates, mainly glucose that comes from the starch degradation during technological processes. The degree of the protein glycation has an effect on the quality of malt and beer and on the properties of the beer foam. A combination of two-dimensional high performance liquid chromatography (2D-HPLC) and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF MS) was used for the analysis of the protein extracts that were reduced, alkylated, and degraded enzymatically without prior protein separation. This so-called "shot-gun" approach enabled us to determine glycation sites in one third of the proteins identified in the study and to propose potential glycation markers for fast and efficient monitoring during malting.

Investigation of thermal denaturation of barley nonspecific lipid transfer protein 1 (ns-LTP1b) by nuclear magnetic resonance and differential scanning calorimetry.

The process of thermal denaturation of a covalently modified form of barley grain nonspecific lipid transfer protein 1b (ns-LTP1b) was investigated by nuclear magnetic resonance (NMR) and differential scanning calorimetry up to 115 degrees C. The denaturation was found to be irreversible and highly cooperative. A method of numerical quantitative analysis allowing us to fit the NMR data to a transition state model without further simplification was developed. On the basis of the obtained values of transition state enthalpy and entropy, the rate of denaturation was calculated as a simple measure of protein stability at various temperatures. The effect of disulfide bond reduction on thermal denaturation of ns-LTP1b was studied and discussed in the context of quality control of barley products during storage and processing.

Influence of different proteomic protocols on degree of high-coverage identification of nonspecific lipid transfer protein 1 modified during malting.

Both top-down (combining protein separation with MS analysis of intact proteins) and bottom-up (MS analysis of digested proteins) proteomic approaches were used for detailed characterization of nonspecific lipid transfer protein from barley malt. The aim was obtaining high-coverage of the primary structure of the proteins and the determination of PTMs such as lipid adduction and glycation. Here we present an influence of 15 proteomic protocols (differing in applied separation technique, enzyme and digestion procedure) on the extent of the coverage of the protein primary structure. The most successful protocols were in-gel digestion with trypsin of alkylated protein and in-solution digestions with trypsin or trypsin/chymotrypsin mixture of the nonalkylated protein. Totally, full sequence coverage based on the PMF and 85% sequence coverage based on the peptide fragmentation including PTMs was obtained.

Detection of hydrolysis of lipid post-translational modifications during gel-electrophoresis-based proteomic protocol.

The influence of sample preparation on the identification of a lipid PTM was examined. Nonspecific lipid transfer protein 1 (LTP1) from barley is modified with a lipid-like molecule of mass of 294 Da. This modification was detected in the MS analysis of intact protein samples but no lipid-bound peptide was observed in the MS analysis of the in-gel digested LTP1 after an SDS-PAGE separation of the protein mixture. By using SEC instead of SDS-PAGE, the lipid-modified peptide was observed after in-solution enzymatic digestion of the SEC fraction containing LTP1. Conditions of individual steps of the gel-electrophoresis-based protocol were tested to find their effect on the removal of the lipid PTM from LTP1. The influences of particular solutions used in the gel-electrophoresis-based protocol on the hydrolysis of lipids were investigated. It was found that denaturing conditions, in combination with alkaline pH, have a major influence on the hydrolysis of the ester bond. Especially, the electrophoretic buffer has a strong influence on the hydrolysis of the lipid PTM (in the intact molecule) of LTP1.

Determination of carbohydrates in juices by capillary electrophoresis, high-performance liquid chromatography, and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry.

The objective of this work was to develop a sample preparation procedure for determination of the carbohydrate profiles in commercial juice samples by three principally different analytical methods: capillary electrophoresis (CE) with indirect detection, high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The preparation and purification of juice samples prior to analysis is described. The method using Carrez reagents was found to be an efficient preparation tool for all three methods. The addition of Carrez reagents to the samples for mass analysis improved the quality of the mass spectra of oligosaccharides. The amounts of glucose, fructose, and sucrose as major carbohydrates in fruit juices measured by CE using a simple instrument are in good agreement with the HPLC values and the data declared by the producers of the juices. The results from both methods are critically evaluated and their impact for studies of authenticity is discussed. The decrease of sucrose amount during the storage of samples was explained by acid hydrolysis of this disaccharide.