The isotopic exchange of oxygen as a tool for detection of the glycation sites in proteins

A nonenzymatic reaction of reducing sugars with the free amino group located at the N terminus of the polypeptide chain or in the lysine side chain results in glycation of proteins. The fragments of glycated proteins obtained by enzymatic hydrolysis could be considered as the biomarkers of both the aging process and diabetes mellitus. Here we propose a new method for the identification of peptide-derived Amadori products in the enzymatic digest of glycated proteins. The products of enzymatic hydrolysis of the model protein ubiquitin were incubated with H₂¹⁸O under microwave activation. We observed that at these conditions the Amadori compounds selectively exchange one oxygen atom in the hexose moiety. The characteristic isotopic pattern of Amadori products treated with H₂¹⁸O allows fast and convenient identification of this group of compounds, whereas nonglycated peptides are not susceptible to isotopic exchange.     

Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome

The current progression from genomics to proteomics is fueled by the realization that many properties of proteins (e.g., interactions, post-translational modifications) cannot be predicted from DNA sequence. Although it has become feasible to rapidly identify proteins from crude cell extracts using mass spectrometry after two-dimensional electrophoretic separation, it can be difficult to elucidate low-abundance proteins of interest in the presence of a large excess of relatively abundant proteins. Therefore, for effective proteome analysis it becomes critical to enrich the sample to be analyzed in subfractions of interest. For example, the analysis of protein kinase substrates can be greatly enhanced by enriching the sample of phosphorylated proteins. Although enrichment of phosphotyrosine-containing proteins has been achieved through the use of high-affinity anti-phosphotyrosine antibodies, the enrichment of phosphoserine/threonine-containing proteins has not been routinely possible. Here, we describe a method for enriching phosphoserine/threonine-containing proteins from crude cell extracts, and for subsequently identifying the phosphoproteins and sites of phosphorylation. The method, which involves chemical replacement of the phosphate moieties by affinity tags, should be of widespread utility for defining signaling pathways and control mechanisms that involve phosphorylation or dephosphorylation of serine/threonine residues.     

Proteome analysis of differentially displayed proteins as a tool for the investigation of symbiosis

Two-dimensional gel electrophoresis was used to identify differentially displayed proteins expressed during the symbiotic interaction between the bacterium Sinorhizobium meliloti strain 1021 and the legume Melilotus alba (white sweetclover). Our aim was to characterize novel symbiosis proteins and to determine how the two symbiotic partners alter their respective metabolisms as part of the interaction, by identifying gene products that are differentially present between the symbiotic and non-symbiotic states. Proteome maps from control M. alba roots, wild-type nodules, cultured S. meliloti, and S. meliloti bacteroids were generated and compared. Over 250 proteins were induced or up-regulated in the nodule, compared with the root, and over 350 proteins were down-regulated in the bacteroid form of the rhizobia, compared with cultured cells. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprint analysis, in conjunction with data base searching, were used to assign putative identity to nearly 100 nodule, bacterial, and bacteroid proteins. These included the previously identified nodule proteins leghemoglobin and NifH as well as proteins involved in carbon and nitrogen metabolism in S. meliloti. Bacteroid cells showed down-regulation of several proteins involved in nitrogen acquisition, including glutamine synthetase, urease, a urea-amide binding protein, and a PII isoform, indicating that the bacteroids were nitrogen proficient. The down-regulation of several enzymes involved in polyhydroxybutyrate synthesis and a cell division protein was also observed. This work shows that proteome analysis will be a useful strategy to link sequence information and functional genomics.     

Kinetic modeling as a tool to understand the influence of cell culture process parameters on the glycation of monoclonal antibody biotherapeutics

Glycation, the nonenzymatic reaction between the reducing sugar glucose and the primary amine residues on amino acid side chains, commonly occurs in the cell culture supernatant during production of therapeutic monoclonal antibodies (mAbs). While glycation has the potential to impact efficacy and pharmacokinetic properties for mAbs, the most common undesirable impact of glycation is on the distribution of charged species, often a release specification for commercial processes. Existing empirical approaches are usually insufficient to rationalize the effects of cell line and process changes on glycation. To address this gap, we developed a kinetic model for estimating mAb glycation levels during the cell culture process. The rate constant for glycation, including temperature and pH dependence, was estimated by fitting the kinetic model to time-course glycation data from bioreactors operated at different process settings that yielded a wide range of glycation values. The parameter values were further validated by independently estimating glycation rate constants using cell-free incubation studies at various temperatures. The model was applied to another mAb, by re-estimating the activation energy to account for effect of a glycation “hotspot”. The model was further utilized to study the role of temperature shift as an approach to reduce glycation levels in the manufacturing process for mAb2. While a downshift in temperature resulted in lowering of glycation levels for mAb2, the model helped elucidate that this effect was caused due to contribution from changes in glucose consumption, mAb secretion and temperature, instead of a direct impact of temperature alone on the kinetic rate of glycation.     

GFP as a tool for the analysis of proteins in the flagellar basal apparatus of Chlamydomonas

Green fluorescent protein (GFP) was used to analyse three proteins in the flagellar basal apparatus of C. reinhardtii: (1) Striated fiber assemblin (SFA), the major component of the striated microtubule-associated fibers; (2) Centrin, present in the nucleus basal body connectors (NBBCs) and the distal connecting fiber (dCF) between the two basal bodies; and (3) DIP13, the Chlamydomonas homologue of human autoantigen NA14. The fusions co-localized with the wild-type proteins when expressed moderately. Overexpression of centrin-GFP and DIP13-GFP resulted in the formation of large aggregates and disturbed the distribution of the respective wild-type proteins. The amount of wild-type DIP13 was significantly reduced in cells overexpressing DIP13-GFP. Moreover, the cells frequently failed to assemble full-length flagella and flagellar regeneration was delayed, indicating a role of DIP13 during flagellar assembly. In contrast, overexpression of GFP-SFA, which retained more wild-type properties than SFA-GFP, increased the size of the striated fibers without altering the cross-shaped pattern. Abnormal patterns were observed in centrin-deficient cells, suggesting that centrin is required for proper localization of SFA. Photobleaching of GFP-SFA fibers indicated that GFP-SFA in the fibers is turned over slowly. Conditionally expressed centrin-GFP was incorporated into NBBCs in regions close to the basal bodies, but underrepresented in the dCF, indicative of a different dynamic of these two centrin fibers. Bending of the NBBCs was observed in vivo during flagellar motion, indicating that the filaments are flexible. In conclusion, in Chlamydomonas GFP-tagging is a useful tool for yielding new insights into the function and properties of the analyzed proteins.     

Anglers' records as a tool for assessing changes in fish populations

Published anglers’ records from Polish rivers between 1966–1991 were used to show shifts in body weight of two obligatory riverine species: barbel [Barbus barbus(L.)], and chub [Leuciscus cephalus (L.)]. The body weight of barbel significantly decreased while that of chub did not. In 1966–89, the quality of inland waters continuously decreased, a result mainly from nutrient element input (domestic and agricultural). Hence, we consider two factors which were mainly responsible for reduction in fish size: overfishing and, perhaps, eutrophication.     

Optical trap as a tool for studying motor proteins

The review briefs the optical trap method, a modern experimental tool based on the recently discovered ability of light to trap and hold micron and submicron particles in a focused beam. The physical principle underlying the optical trap and the opportunities that it provides for studying the molecular nature of biological motility are considered. Several studies into the physical characteristics and functions of single motor protein molecules performed using the optical trap and recording nanometer displacements and piconewton forces are analyzed.     

Receptor-Galpha fusion proteins as a tool for ligand screening

We have prepared fusion proteins of muscarinic M1-M5 receptors with alpha subunits of G proteins Gi1, Gi2, Gs, G11, G16 and chimera of G protein alpha subunits using the bacurovirus-Sf9 expression system. In fusion proteins such as M2-Gi1alpha and M4-Gi1alpha, agonist caused the decrease in the apparent affinity for GDP of these fusion proteins and then the increase in [35S]GTPgammaS binding in the presence of GDP. Thus we could use the membrane preparation expressing these fusion proteins as a tool to screen agonists and antagonists. On the other hand, the effect of agonists to decrease the apparent affinity for GDP was not clearly observed in fusion proteins of Gq/G11-coupled receptors such as M1-G11alpha, M3-G11alpha, and M5-G11alpha. The effect of agonists could be observed for fusion proteins with G16alpha of muscarinic M1, M2 and adrenergic beta2 receptors, but the extent of the effect was much less than that for fusion proteins with Gi1alpha of Gi/Go-coupled receptors. Fusion proteins of M1 receptors with Gi1alpha or chimera of G16alpha and Gi2alpha were also not effective in detecting the action of agonists.     

Free nonimmobilized ligands as a tool for purification of proteins

Purification of proteins on a large scale is a complex multistep process, and alternative economic strategies are required. This study presents a novel approach (Affinity Sinking, AS) for purification of native proteins utilizing nonimmobilized modified ligands. The nonimmobilized state of the ligand circumvents the need for immobilizing ligands to polymeric supports. Therefore, purification from large volumes can be accomplished without the use of industrial-scale affinity columns. The mechanism of product capture is formation and precipitation of a specific [target-protein/modified-ligand] complex by using a soluble interconnecting entity that generates an insoluble [target-protein/modified-ligand/interconnecting entity] sediment containing the target protein. Rabbit IgG and two glycoproteins were purified accordingly, utilizing free avidin (as the interconnecting entity) and either desthiobiotinylated-protein A (DB-ProA) or desthiobiotinylated-concanavalin A (DB-ConA) as the modified ligand. The recovery yields for the IgG and the two glycoproteins were 80-86% and 70-75%, respectively. Target proteins are eluted from the generated pellet nearly without disrupting the [modified-ligand/interconnecting entity] macro-complex, thus enabling a practical procedure of recovering target proteins. Leaching of the DB-ProA ligand under eluting conditions (pH 3) was found to be lower than 1%. The two modified ligands, DB-ProA and DB-ConA, were regenerated without any chromatographic procedure in 80% and 85%-89% yield, respectively. The advantage of excluding the polymeric component from the purification process and obtaining highly purified proteins has been demonstrated, and it implies that other contaminants (e.g. endotoxins, prions, host DNA) could be excluded as well, thereby reducing the number of purification steps in a typical downstream process.     

Protein-protein interactions as a tool for site-specific labeling of proteins

Probing structures and dynamics within biomolecules using ensemble and single-molecule fluorescence resonance energy transfer requires the conjugation of fluorophores to proteins in a site-specific and thermodynamically nonperturbative fashion. Using single-molecule fluorescence-aided molecular sorting and the chymotrypsin inhibitor 2-subtilisin BPN' complex as an example, we demonstrate that protein-protein interactions can be exploited to afford site-specific labeling of a recombinant double-cysteine variant of CI2 without the need for extensive and time-consuming chromatography. The use of protein-protein interactions for site-specific labeling of proteins is compatible with and complementary to existing chemistries for selective labeling of N-terminal cysteines, and could be extended to label multiple positions within a given polypeptide chain.     

Remote sensing as a tool for assessing water quality in Loosdrecht lakes

The underwater light field in 7 lakes in the Loosdrecht lake area was measured in situ. Subsurface upwelling irradiance and irradiance reflectance, together with estimations of scattering and laboratory measurements of absorption by aquatic humus and particulate matter, enabled an analysis of the spectral signature of these waters. Aircraft imaging spectrometer measurements of upwelling radiance at 1 km altitude were used to simulate the PMI Chlorophyll #1, the CAESAR Inland Water Mode spectral bandsets and the Thematic Mapper bands 1 to 4. This made it possible to compare the effects of spectral band width and selection on the estimation of water quality parameters. Correlations increased to r > 0.94, at a significance level of 1% for the simulated C-IWM data with the 6 water quality parameters. Images of the PMI Chlorophyll #1 and of the TM were analysed and found to be in accordance with the statistical modelling results.A significant increase in correlation of remote sensing data with water quality parameters can be achieved through the selective use of 10 to 20 nm wide bands in the spectral range of 500 to 720 nm in these eutrophic waters. Sum of chlorophyll a and phaeopigments, seston dry weight, Secchi disc transparency, and coefficients for vertical attenuation of light, absorption and scattering can be estimated accurately. TM image data for water quality assessment is of limited use due to the relatively low spectral and radiometric resolution. However, the revisit capability and relatively low price per area are positive aspects of these satellite images.Abbreviations CAESAR = CCD Airborne Experimental Scanner for Applications in Remote sensing - C-IWM = CAESAR Inland Water Mode - CCD = charge coupled device - EOS-A = Earth Observation System Platform A - PAR = photosynthetically active radiation from 400–700 nm. - PMI = Programmable Multispectral Imager - RSLL = Remote Sensing Loosdrecht Lakes Project - SPOT = Systeme Pour l'Observation de la Terre - SPOT-HRV = Sensor on board of the SPOT satellite - TM = Thematic Mapper instrument aboard the Landsat 5 satellite     

Bile analysis as a tool for assessing integrity of biliary epithelial cells after cold ischemia--reperfusion of rat livers

Previous morphological studies failed to show appreciable injury of biliary epithelial cells (BEC) after cold ischemia of rat liver, although recent evidence indicated that BEC integrity and function were impaired in this model. We tested the hypothesis that analysis of bile for enzymes, such as lactate dehydrogenase (LDH), alanine transaminase (ALT), and aspartate transaminase (AST), can be used for assessing cold ischemic injury of BEC. Furthermore, we examined whether biliary gamma-glutamyltransferase (GGT) reflects warm ischemic injury of BEC and whether normothermic reperfusion aggravates the negative effect of cold ischemia on BEC integrity and function. Rat livers were reperfused after different periods of cold or warm ischemia using a blood-free perfusion model. Compared with controls, perfusate LDH, ALT, and AST levels and parameters of hepatocyte function, including hepatocyte tight junction permeability, were not significantly altered by 18-h cold ischemia. On the other hand, 9-h cold ischemia markedly increased biliary LDH, ALT, and AST levels. However, only LDH release into the bile was strongly dependent on the time of cold storage. Biliary GGT, LDH, and glucose levels decreased during the reperfusion period following 18-h cold ischemia. The results suggest that biliary LDH can be used for assessing injury of BEC in cold-preserved livers and that normothermic reperfusion does not aggravate preservation-induced injury of BEC after cold ischemic storage.     

Capillary electrophoresis-mass spectrometry as a characterization tool for therapeutic proteins

With the increasing use of capillary electrophoresis (CE) in the biotechnology industry, there is a demand for analytical tools and methodology that can be used to characterize CE profiles. This article describes the implementation and optimization of a robust online CE-mass spectrometry (CE-MS) system used for the characterization of several CE assays developed at Genentech Inc. These assays include CE as a complement to reverse-phase peptide mapping for the identification of small peptides eluting in the void volume, profiling N-linked glycopeptide heterogeneity, and determining O-linked site occupancy. In addition, CE-MS was used to confirm major 8-aminopyrene-1,3,6-trisulfonate (APTS)-labeled glycans released from recombinant antibodies that are routinely profiled by CE-laser-induced fluorescence (CE-LIF). For each study, CE-MS was able to successfully identify components seen in UV or LIF electropherograms, thereby expanding the capability of CE and CE-MS for profiling biomolecules.     

Caenorhabditis elegans as a screening tool for the endothelial cell-derived putative aging-related proteins detected by proteomic analysis

Endothelial cells go through progressive pathophysiologic modification as cellular senescence progresses. In vitro, endothelial cell senescence is accompanied by failure of proliferation and by perturbations in gene and protein expressions. Moreover, this cellular senescence in culture has been proposed to reflect processes that occur in the organism in vivo and free radical theory is accepted to be the most plausible explanation for this process. We have screened proteins involved in both cellular senescence and reactive oxygen species induced condition using 2-D gel analysis and found that ubiquitin carboxyl terminal hydrolase L1, peroxyredoxin 2, peroxyredoxin 4, fatty acid binding proteins (FABPs), and 5'-AMP-activated protein kinase beta-1 subunit were candidate aging-related proteins. To evaluate in vivo function of these proteins, Caenorhabditis elegans (C. elegans) knock-down system using RNA interference was applied. Aging-specific expression of lipofucsin and the lifespan of knocked-down C. elegans were observed to assess the outcome. Interestingly, the inhibition of the genes led to short lifespan and earlier accumulation of lipofucsin with increasing age when compared with the wild type. These results suggest that the above genes may be related to cellular senescence process in determining the longevity in C. elegans and that gene inactivation renders animals susceptible to oxidative stress.     

Lipid-impregnated filters as a tool for studying the electric current-generating proteins.

Porous filters and collodion film impregnated with decane solution of phospholipids, were used for measurements of electric potential differences generated by bacteriorhodopsin, chromatophore redox chain, H⁺-ATPase, pyrophosphatase, and mitochondrial respiratory chain. It was shown that reconstituted proteoliposomes, containing e.g., bacteriorhodopsin or natural coupling membrane vesicles, such as Rhodospirillum rubrum chromatophores, can be attached to a filter surface by means of Ca²⁺ or Mg²⁺ ions. Addition of the respective energy source was found to result in electric potential difference being generated across the filter. This effect was measured directly by $\text{Ag}\text{AgCl}$ electrodes immersed into electrolyte solutions on both sides of the filter. Using a phospholipid-impregnated collodion film one can measure electric responses as fast as 300 nsec. The phospholipid-impregnated filters turned out to be sensitive and reliable electrodes for measuring the concentration of synthetic penetrating ions, such as phenyldicarbaundecaborane, tetraphenylborate, tetrapentylammonium, and tetraphenylphosphonium. By measuring changes in the concentration of these ions in the suspension of proteoliposomes, chromatophores, mitochondria, or bacterial cells, one can follow the formation and dissipation of transmembrane potential differences in these systems. It is shown that the phospholipid-impregnated filters are much more reliable and handy than planar phospholipid membranes previously used for studying electrogenic activity of electric current-producing membrane proteins.     

Anti-sulfonylbenzoate antibodies as a tool for the detection of nucleotide-binding proteins for functional proteomics

Proteins that bind ATP and GTP are important cellular components. We developed an immunological approach to selectively tag nucleotide-binding proteins based on the use of 5'-[4-(fluorosulfonyl)benzoyl]adenosine and 5'-[4-(fluorosulfonyl)benzoyl]guanosine affinity tags and an antibody against 4-(sulfonyl)benzoate. Detection follows affinity labeling, gel electrophoresis, and ester bond cleavage to expose the epitope. Trial analyses of labeled proteins from lymphoid cells identified multiple ATP-binding proteins, including chaperones, actin, kinases, an RNA splicing factor, a membrane ATPase, and ATP synthase.     

NMR as a tool for elucidating the structures of circular and knotted proteins

Cyclotides are a recently discovered family of mini-proteins that have a head-to-tail cyclised backbone stabilized by a knotted arrangement of three disulfide bonds. They have a wide range of biological activities, including uterotonic, anti-bacterial, anti-HIV, and anti-tumour activity but their insecticidal activities suggest that their natural function is in plant defense. They are exceptionally resistant to chemical, enzymatic and thermal treatments because of their unique structural scaffold. This stability and resistance to proteolysis makes them a potentially valuable protein engineering tool at the interface of chemistry and biology: they have the structure of proteins but the stability and biophysical properties of organic molecules. In this review the role of NMR in defining the structures of cyclotides is described.     

Procrustes analysis as a tool for land management

Generalized Procrustes analysis (GPA) is a multivariate technique that involves transformations of data matrices to provide optimal comparability. We propose GPA to quantify the concordance among sets of variables that characterize natural, human and productive subsystems. When the land use fits in with the physical support of agricultural production, people's well-being should be evident in a high concordance between the land use and the social conditions. In a situation of instability each set of variables operates in diverse directions resulting in lower resilience and sustainability. Two GPA were performed, between physical support and land use data sets (concordance = 67.4%), and between land use and social conditions data sets (concordance = 65.3%). The interplay between the pair of concordance values constitutes a bi-dimensional index which serves as an ecological indicator. Based on bootstrap confidence interval, the 49 counties of the Pampa Ecoregion, Argentina, were classified in medium, high or low concordance. The lack of concordance is an indicator of imbalances which may contribute to guide environmental management.