Radovan Hynek

Classification of protein binders in artist's paints by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry: an evaluation of principal component analysis (PCA) and soft independent modelling of class analogy (SIMCA)

Proteomics techniques are increasingly applied for the identification of protein binders in historical paints. The complex nature of paint samples, with different kinds of pigments mixed into, and degradation by long term exposure to light, humidity and temperature variations, requires solid analysis and interpretation methods. In this study matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectra of tryptic-digested paint replicas are subjected to principal component analysis (PCA) and soft independent modelling of class analogy (SIMCA) in order to distinguish proteinaceous binders based on animal glues, egg white, egg yolk and milk casein from each other. The most meaningful peptide peaks for a given protein class will be determined, and if possible, annotated with their corresponding amino acid sequence. The methodology was subsequently applied on egg temperas, as well as on animal glues from different species. In the latter small differences in the MALDI-TOF mass spectra can allow the determination of a mammal or sturgeon origin of the glue. Finally, paint samples from the 16(th) century altarpiece of St Margaret of Antioch (Mlynica, Slovakia) were analysed. Several expected peaks are either present in lower abundance or completely missing in these natural aged paints, due to degradation of the paints. In spite of this mammalian glue was identified in the St Margaret samples.

Plant PIP2-dependent phospholipase D activity is regulated by phosphorylation

Phospholipase D (PLD) forms the major family of phospholipases that was first discovered and cloned in plants. In this report we have shown, for the first time, that C2 phosphatidylinositol‐4,5‐bisphosphate (PIP2)‐dependent PLD(s) from 5 day hypocotyls of Brassica oleracea associated with plasma membrane is covalently modified‐phosphorylated. Pre‐incubation of the plasma membrane fraction with acid phosphatase resulted in concentration‐dependent inhibition of PIP2‐dependent PLD activity. Using matrix‐assisted laser desorption/ionization time of flight mass spectrometry of tryptic in‐gel digests, the BoPLDγ1,2 isoform was identified. Comparing the spectra of the proteins obtained from the plasma membrane fractions treated and non‐treated with acid phosphatase, three peptides differing in the mass of the phosphate group (80 Da) were revealed: TMQMMYQTIYK, EVADGTVSVYNSPR and KASKSRGLGK which possess five potential Ser/Thr phosphorylation sites. Our findings suggest that a phosphorylation/dephosphorylation mechanism may be involved in the regulation of plant PIP2‐dependent PLDγ activity.

Assessment of green cleaning effectiveness on polychrome surfaces by MALDI‐TOF mass spectrometry and microscopic imaging

This article proposes an innovative methodology which employs nondestructive techniques to assess the effectiveness of new formulations based on ionic liquids, as alternative solvents for enzymes (proteases), for the removal of proteinaceous materials from painted surfaces during restoration treatments. Ionic liquids (ILs), also known as “designer” solvents, because of their peculiar properties which can be adjusted by selecting different cation‐anion combinations, are potentially green solvents due totheir low vapour pressure. In this study, two ionic liquids were selected: IL1 (1‐butyl‐3‐methylimidazolium tetrafluoroborate ([BMIM][BF4])) and IL2 (1‐ethyl‐3‐methylimidazolium ethylsulphate ([EMIM][EtSO4])). New formulations were prepared with these ILs and two different proteases (E): one acid (E1—pepsin) and one alkaline (E2—obtained from Aspergillus sojae). These formulations were tested on tempera and oil mock‐up samples, prepared in accordance with historically documented recipes, and covered with two different types of protein‐based varnishes (egg white and isinglass—fish glue). A noninvasive multiscale imaging methodology was applied before and after the treatment to evaluate the cleanings effectiveness. Different microscopic techniques—optical microscopy (OM) with visible and fluorescent light, scanning electron microscopy (SEM) and atomic force microscopy (AFM)—together with Matrix‐Assisted Laser Desorption/Ionization—Time of Flight Mass Spectrometry (MALDI‐TOF MS) were applied on areas cleaned with the new formulations (IL + E) and reference areas cleaned only with the commercial enzyme formulations (gels). MALDI‐TOF proved particularly very useful for comparing the diversity and abundance of peptides released by using different enzymatic systems. Microsc. Res. Tech. 77:574–585, 2014.

Proteomic Response of Hordeum vulgare cv. Tadmor and Hordeum marinum to Salinity Stress: Similarities and Differences between a Glycophyte and a Halophyte.

Response to a high salinity treatment of 300 mM NaCl was studied in a cultivated barley Hordeum vulgare Syrian cultivar Tadmor and in a halophytic wild barley H. marinum. Differential salinity tolerance of H. marinum and H. vulgare is underlied by qualitative and quantitative differences in proteins involved in a variety of biological processes. The major aim was to identify proteins underlying differential salinity tolerance between the two barley species. Analyses of plant water content, osmotic potential and accumulation of proline and dehydrin proteins under high salinity revealed a relatively higher water saturation deficit in H. marinum than in H. vulgare while H. vulgare had lower osmotic potential corresponding with high levels of proline and dehydrins. Analysis of proteins soluble upon boiling isolated from control and salt-treated crown tissues revealed similarities as well as differences between H. marinum and H. vulgare. The similar salinity responses of both barley species lie in enhanced levels of stress-protective proteins such as defense-related proteins from late-embryogenesis abundant family, several chaperones from heat shock protein family, and others such as GrpE. However, there have also been found significant differences between H. marinum and H. vulgare salinity response indicating an active stress acclimation in H. marinum while stress damage in H. vulgare. An active acclimation to high salinity in H. marinum is underlined by enhanced levels of several stress-responsive transcription factors from basic leucine zipper and nascent polypeptide-associated complex families. In salt-treated H. marinum, enhanced levels of proteins involved in energy metabolism such as glycolysis, ATP metabolism, and photosynthesis-related proteins indicate an active acclimation to enhanced energy requirements during an establishment of novel plant homeostasis. In contrast, changes at proteome level in salt-treated H. vulgare indicate plant tissue damage as revealed by enhanced levels of proteins involved in proteasome-dependent protein degradation and proteins related to apoptosis. The results of proteomic analysis clearly indicate differential responses to high salinity and provide more profound insight into biological mechanisms underlying salinity response between two barley species with contrasting salinity tolerance.

Differentiation of Cronobacter spp. by tryptic digestion of the cell suspension followed by MALDI-TOF MS analysis

Intact cell MALDI-TOF mass spectrometry is a rapid tool for the identification and classification of microorganisms, now widely used even in clinical laboratories. However, its distinctive power is not sufficient for some closely-related species. The genus Cronobacter, formerly known as Enterobacter sakazakii, contains such species. In this work, a new method for the differentiation of five Cronobacter species is presented involving the tryptic digestion of cytoplasmatic proteins followed by MALDI mass spectrometry analysis. A database was developed for use in Bruker Biotyper software including 52 reference spectra and tested on a set of 45 samples with an overall accuracy of about 80%. The possibility of measurement automation and the short time and low cost requirements of this method compared to those of biochemical tests or PCR methods make it a supplementary option to intact cell MALDI, providing additional information about the differentiation of problematic species.

Expression and purification of myristoylated matrix protein of Mason-Pfizer monkey virus for NMR and MS measurements

Matrix proteins play multiple roles both in early and late stages of the viral replication cycle. Their N-terminal myristoylation is important for interaction with the host cell membrane during virus budding. We used Escherichia coli, carrying N-myristoyltransferase gene, for the expression of the myristoylated His-tagged matrix protein of Mason-Pfizer monkey virus. An efficient, single-step purification procedure eliminating all contaminating proteins including, importantly, the non-myristoylated matrix protein was designed. The comparison of NMR spectra of matrix protein with its myristoylated form revealed substantial structural changes induced by this fatty acid modification.

High‐throughput workflow for identification of phosphorylated peptides by LC‐MALDI‐TOF/TOF‐MS coupled to in situ enrichment on MALDI plates functionalized by ion landing

We report an MS-based workflow for identification of phosphorylated peptides from trypsinized protein mixtures and cell lysates that is suitable for high-throughput sample analysis. The workflow is based on an in situ enrichment on matrix-assisted laser desorption/ionization (MALDI) plates that were functionalized by TiO2 using automated ion landing apparatus that can operate unsupervised. The MALDI plate can be functionalized by TiO2 into any array of predefined geometry (here, 96 positions for samples and 24 for mass calibration standards) made compatible with a standard MALDI spotter and coupled with high-performance liquid chromatography. The in situ MALDI plate enrichment was compared with a standard precolumn-based separation and achieved comparable or better results than the standard method. The performance of this new workflow was demonstrated on a model mixture of proteins as well as on Jurkat cells lysates. The method showed improved signal-to-noise ratio in a single MS spectrum, which resulted in better identification by MS/MS and a subsequent database search. Using the workflow, we also found specific phosphorylations in Jurkat cells that were nonspecifically activated by phorbol 12-myristate 13-acetate. These phosphorylations concerned the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and its targets and were in agreement with the current knowledge of this signaling cascade. Control sample of non-activated cells was devoid of these phosphorylations. Overall, the presented analytical workflow is able to detect dynamic phosphorylation events in minimally processed mammalian cells while using only a short high-performance liquid chromatography gradient.

Influence of phosphorylation on proteolytic cleavage

ABSTRACT Phosphorylation of an amino acid residue close to the peptide bond, which is a target of a specific proteolytic enzyme, seems to cause inhibition of such a cleavage. This finding may have a number of important consequences. The effect of phosphorylation of a model peptide on its proteolytic cleavage by trypsin was studied. The velocity of peptide bond cleavage of nonphosphorylated synthetic peptide was compared with cleavage velocity of the same bond of synthetic peptide phosphorylated on serine residue located in closed proximity of the specifically cleaved peptide bond. It was shown that the enzymatic cleavage was inhibited by phosphorylation of mentioned amino acid residue. Reversed-phase high-performance chromatography (RP-HPLC) was used for the fast and reliable analysis of reaction mixtures. Influence of the addition of alkaline phosphatase on proteolytical cleavage of casein was also investigated. RP-HPLC peptide maps of β-casein cleaved by trypsin in the presence and absence of alkaline phosphatase were compared. It was proven that the action of acid phosphatase affects the composition of the resulting proteolytic digest.

IMPROVED APPROACH FOR THE LABELING OF ARGININE, GLUTAMIC, AND ASPARTIC ACID SIDE CHAINS IN PROTEINS USING CHROMATOGRAPHIC TECHNIQUES

Specific chemical modification is one of the basic techniques of protein chemistry. Inter alia can be used for detection of surface accessible amino acid residues; this information is of particular importance for studies of the participation of residues in intermolecular interactions of a protein. We achieved an improvement of the technique for arginine, aspartic, and glutamic acid modification using a simple combination of gel permeation and reversed-phase chromatography prior to mass spectrometry analysis. The improved protocol was tested on cytochrome c and M-PMV matrix protein. In both proteins, all accessible arginines and a high number of acidic amino acids were modified. These results indicate that the new protocol can be useful in protein structure analysis, generally.

Disulfide bond decay during matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry experiments

In our laboratory, we have been studying the reductive processes that occur during matrix-assisted laser desorption/ionization (MALDI) experiments. Recently, we have finished an analysis of the DHB matrix effect on the azo group in cyclic peptides. However, deep understanding of disulfide bond behaviour during a mass spectrometry (MS) experiment is much more important in proteomics as its reduction can cause serious errors in protein spectra interpretation. Therefore, we have focused on intra- and intermolecular disulfide bonds as well as disulfide bonds connecting cysteine and 2-thio-5-nitrobenzoic acid (TNB, Ellmans reagent modification) in model peptides during MALDI MS measurements. While the reduction was not observed for intra- and intermolecular cysteine-cysteine disulfide bonds, the disulfide connection between cysteine and TNB was always affected. It was proved that TNB and Ellmans reagent can act as a matrix itself. The results obtained enabled us to propose a reaction mechanism model which is able to describe the phenomena observed during the desorption/ionization process of disulfide-containing molecules.