Conductive carbon tape as a sample platform for microwave-based MALDI MS detection of proteins and phosphoproteins

In this study, we developed a novel microwave-assisted protein preparation and digestion method for matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry analysis and identification of proteins that involves using conductive carbon tape as a sample platform for sample preparation (reduction and alkylation) and digestion under microwave heating and as a plate for MALDI analysis. This method allows for the enzymatic digestion products of proteins to be directly analyzed by MALDI mass spectrometry and results in a marked reduction in sample loss. Our protocol requires only a small volume (1 μL) of reaction solvent, which increases the frequency of enzyme-to-protein contact, thereby resulting in more efficient digestion of sample than conventional in-solution digestion methods. To test this protocol, we used magnetic iron (II, III) oxide nanoparticles as concentrating probes to enrich phosphopeptides from a mixture of peptides in enzymatically digested protein samples. We found that the one-pot on-tape-based protein preparation and digestion under microwave heating combined with the on-tape-based enrichment method not only dramatically reduced the time required for phosphopeptides analysis but also allowed for the simultaneous identification of phosphoproteins. The advantages of our protocol include ease of use, high digestion efficiency, high specificity, and rapid (15 min) identification of proteins and enrichment of phosphopeptides in a mixture of enzymatically digested protein samples.     

Vortex-assisted tryptic digestion

The effect of vortex‐induced vibration during tryptic digestion was investigated by applying different vibrational speeds (0, 600, 1200, or 2500 rpm) to digestion solutions for varying durations (10, 20, 30, 40, or 60 min) at two different incubation temperatures (25°C or 37°C). The most rapid digestion was observed with the highest vibrational speed and temperature. With the application of 2500 rpm at 37°C, the tryptic digestion of each of three standard proteins (cytochrome c, myoglobin, or bovine serum albumin) provided complete disappearance of the protein within 60 min, as determined by matrix‐assisted laser desorption/ionization mass spectrometry. Compared to conventional overnight digestion, 60‐min vortex‐assisted tryptic digestion generated longer peptides, due primarily to the limited digestion time and provided better sequence coverages (89% vs. 78% for cytochrome c, 100% vs. 87% for myoglobin, and 38% vs. 26% for BSA). The longer peptides should be advantageous to analytical methods such as the middle‐down approach that benefit from increased sequence coverage of proteins. Vortex‐assisted tryptic digestion is expected to be a useful method for rapid tryptic digestion of proteins. Copyright © 2010 John Wiley & Sons, Ltd.     

Microwave-assisted enzyme-catalyzed reactions in various solvent systems

The work describes the accelerated enzymatic digestion of several proteins in various solvent systems under microwave irradiation. The tryptic fragments of the proteins were analyzed by matrix-assisted laser desorption/ionization mass spectrometry. Under the influence of rapid microwave heating, these enzymatic reactions can proceed in a solvent such as chloroform, which, under traditional digestion conditions, renders the enzyme inactive. The digestion efficiencies and sequence coverages were increased when the trypsin digestions occurred in acetonitrile-, methanol- and chloroform-containing solutions that were heated under microwave irradiation for 10 min using a commercial microwave applicator. The percentage of the protein digested under microwave irradiation increased with the relative acetonitrile content, but decreased as the methanol content was increased. These observations suggest that acetonitrile does not deactivate the enzyme during the irradiation period; in contrast, methanol does deactivate it. In all cases, the digestion efficiencies under microwave irradiation exceed those under conventional conditions.     

Functional Fe3O4@ZnO magnetic nanoparticle-assisted enrichment and enzymatic digestion of phosphoproteins from saliva

Saliva contains various proteins, particularly abundant are phosphoproteins, that may be related to disease occurrences and that play significant roles in a biological system. Thus, medical diagnostics will benefit tremendously if disease-related protein biomarkers are discovered from saliva. In this paper, we propose and demonstrate an approach using functional zinc oxide coated iron oxide magnetic nanoparticles (Fe₃O₄@ZnO MNPs) as affinity probes to selectively enrich phosphoproteins from complex saliva samples and as microwave absorbers to assist the enrichment and subsequent tryptic digestion of trapped proteins under microwave heating. The target species trapped by MNPs were characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) combined with protein database search. Entire analysis time was shortened to less than 20 min. The detection limit of this approach for a monophosphopeptide was as low as 250 pM (10 μL).     

Rapid and efficient glycoprotein identification through microwave-assisted enzymatic digestion

Identification of protein glycosylation sites is analytically challenging due to the diverse glycan structures associated with a glycoprotein. Mass spectrometry (MS)-based identification and characterization of glycoproteins has been achieved predominantly with the bottom-up approach, which typically involves the enzymatic cleavage of proteins to peptides prior to LC/MS or LC/MS/MS analysis. However, the process can be challenging due to the structural variations and steric hindrance imposed by the attached glycans. Alternatives to conventional heating protocols, that increase the rate of enzymatic cleavage of glycoproteins, may aid in addressing these challenges. An enzymatic digestion of a glycoprotein can be accelerated and made more efficient through microwave-assisted digestion. In this paper, a systematic study was conducted to explore the efficiency of microwave-assisted enzymatic (trypsin) digestion (MAED) of glycoproteins as compared with the conventional method. In addition, the optimum experimental parameters for the digestion such as temperature, reaction time, and microwave radiation power were investigated. It was determined that efficient tryptic digestion of glycoproteins was attained in 15 min, allowing comparable if not better sequence coverage through LC/MS/MS analysis. Optimum tryptic cleavage was achieved at 45°C irrespective of the size and complexity of the glycoprotein. Moreover, MAED allowed the detection and identification of more peptides and subsequently higher sequence coverage for all model glycoprotein. MAED also did not appear to prompt a loss or partial cleavage of the glycan moieties attached to the peptide backbones.     

A nanoporous reactor for efficient proteolysis

A nanoreactor based on mesoporous silicates is described for efficient tryptic digestion of proteins within the mesochannels. Cyano-functionalized mesoporous silicate (CNS), with an average pore diameter of 18 nm, is a good support for trypsin, with rapid in situ digestion of the model proteins, cytochrome c and myoglobin. The generated peptides were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Proteolysis by trypsin-CNS is much more efficient than in-solution digestion, which can be attributed to nanoscopic confinement and concentration enrichment of the substrate within the mesopores. Proteins at concentrations of 2 ng muL(-1) were successfully identified after digestion for 20 min. A biological complex sample extracted from the cytoplasm of human liver tissue was digested by using the CNS-based reactor. Coupled with reverse-phase HPLC and MALDI-TOF MS/MS, 165 proteins were identified after standard protein data searching. This nanoreactor combines the advantages of short digestion time with retention of enzymatic activity, providing a promising way to advance the development of proteomics.     

Trypsin immobilization in ordered porous polymer membranes for effective protein digestion

Fast and effective protein digestion is a vital process for mass spectrometry (MS) based protein analysis. This study introduces a porous polymer membrane enzyme reactor (PPMER) coupled to nanoflow liquid chromatography-tandem MS (nLC-ESI-MS/MS) for on-line digestion and analysis of proteins. Poly (styrene-co-maleic anhydride) (PS-co-MAn) was fabricated by the breath figure method to make a porous polymer membrane in which the MAn group was covalently bound to enzyme. Based on this strategy, microscale PPMER (μPPMER) was constructed for on-line connection with the nLC-ESI-MS/MS system. Its capability for enzymatic digestion with bovine serum albumin (BSA) was evaluated with varied digestion periods. The on-line proteolysis of BSA and subsequent analysis with μPPMER-nLC-ESI-MS/MS revealed that peptide sequence coverage increased from 10.3% (digestion time 10 min) to 89.1% (digestion time 30 min). μPPMER can efficiently digest proteins due to the microscopic confinement effect, showing its potential application in fast protein identification and protease immobilization. Applications of on-line digestion using μPPMER with human plasma and urinary proteome samples showed that the developed on-line method yielded equivalent or better performance in protein coverage and identified more membrane proteins than the in-solution method. This may be due to easy accommodation of hydrophobic membrane proteins within membrane pores.     

Pressure‐assisted tryptic digestion using a syringe

A simple and effective digestion method was developed using a syringe. A 3 mL syringe was used to apply a pressure of 6 atm to expedite tryptic digestion. Application of a pressure of 6 atm during digestion resulted in better digestion efficiency than digestion under atmospheric pressure. The protein peaks in the matrix‐assisted laser desorption/ionization mass spectra of three model proteins (cytochrome c, horse heart myoglobin, and bovine serum albumin (BSA)) completely disappeared within 30 min at 37°C under a pressure of 6 atm, with greater numbers of peptides observed in 30 min pressure‐assisted digestion than in overnight atmospheric pressure digestion. This is mostly due to the miscleaved peptides. Similar sequence coverages were obtained for 30 min pressure‐assisted digestion and overnight atmospheric pressure digestion of the three model proteins (92% vs. 88% for cytochrome c, 100% vs. 97% for horse heart myoglobin, and 53% vs. 53% for BSA). Copyright © 2010 John Wiley & Sons, Ltd.     

Development of an on-line immobilized-enzyme reversed-phase HPLC method for protein digestion and peptide separation

This paper describes use of a novel glass bead-based immobilized-enzyme micro column for simple and swift on-line protein digestion then peptide separation by reversed-phase HPLC. The inexpensive and easily made immobilized-enzyme micro column was prepared from aminopropyl controlled-pore glass that was reacted first with glutaraldehyde then with trypsin in the presence of phosphate buffer. Tryptic digestion of bovine serum albumin (BSA) was achieved simply by passing pretreated protein solution through the laboratory made immobilized-trypsin column; the tryptic fragments were then separated by reversed-phase HPLC. The peptide separation was found to be identical to separation of a sample which had undergone conventional enzymatic protein digestion in solution. Digestion of BSA by the immobilized-trypsin column decreased with increasing flow rate of the solution through the column, and 1.0 μL min⁻¹ was found to be the optimum flow rate for on-line protein digestion with our system. It was also found that the sample required pretreatment with urea before injection, because of a change in the properties of the protein in the presence of urea, and the immobilized-trypsin column lost its function in the presence of acetonitrile. This on-line proteomics system enables simple and rapid protein digestion and was successfully applied to partially micro two-dimensional (2D) chromatographic separation of proteins.     

Glass-chip-based sample preparation and on-chip trypic digestion for matrix-assisted laser desorption/ionization mass spectrometric analysis using a sol-gel/2,5-dihydroxybenzoic acid hybrid matrix

A glass-chip-based sample preparation method for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis of tryptic digests of proteins and intact cells is described. A MALDI matrix, 2,5-dihydroxybenzoic acid (2,5-DHB), was hybridized with sol-gels to generate a sol-gel-derived material. Taking advantage of the characteristics of sol-gels, the sol-gel-derived material readily adhered to the surface of a glass chip through covalent bonding. Only one step of sample preparation, deposition of the sample solution on the glass chip, was required before MALDI-MS analysis. Because 2,5-DHB was homogeneously dispersed on the sol-gel network structure, good spot-to-spot reproducibility was obtained in MALDI analysis using this approach and the analyte signals were uniform throughout the chip. The modified glass chips were robust and effective for at least 1 week. This glass-chip-based matrix preparation method provides a straightforward approach to developing techniques for analyzing the on-chip enzymatic digestion of proteins and intact cells of microorganisms. Cytochrome C and Escherichia coli were used as analytes to demonstrate the feasibility of this approach. The products of the on-chip enzymatic digests were identified through protein database searches.     

Highly efficient enrichment and subsequent digestion of proteins in the mesoporous molecular sieve silicate SBA-15 for matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight/time-of-flight analyzer peptide mapping

Based on a previous study of protein digestion inside the nanoreactor channels of the mesoporous molecular sieve silicate SBA-15 (Chem. Eur. J. 2005, 11: 5391), we have developed a highly efficient enrichment and subsequent tryptic digestion of proteins in SBA-15 for matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight/time-of-flight analyzer (MALDI-TOF/TOF) peptide mapping. The performance of the method is exemplified with myoglobin and cytochrome c. First, protein adsorption isotherms for two standard proteins with a range of initial concentration of proteins were investigated at room temperature. The results revealed that the kinetic adsorption rate of a protein within SBA-15 was independent of initial protein concentration, and a 15-min protein enrichment within SBA-15 could be enough for protein identification in biological samples. It was noticed that no washing steps were needed to avoid protein loss due to desorption from the mesochannels into solution. Second, protein digestion inside the channels of SBA-15 was also optimized. After adsorption of proteins into SBA-15 in 15 min, the trypsin solution (pH 8) was directly added to the SBA-15 beads with immobilized proteins by centrifugation, and then the digestion was performed for 15 min at 37 degrees C. It was observed that a higher peptide sequence covering of 98% for myoglobin was obtained by MALDI-TOF/TOF analysis, compared to in-solution digestion. So the protein digestion inside SBA-15 was proved to be significantly faster and yielded a better sequence coverage. The new procedure allows for rapid protein enrichment and digestion inside SBA-15, and has great potential for protein analysis.     

Rapid characterization of protein chips using microwave-assisted protein tryptic digestion and MALDI mass spectrometry

We demonstrate that the microwave-assisted protein enzymatic digestion (MAPED) method can be successfully applied to the mass spectrometric characterization of proteins captured on the affinity surfaces of protein chips. The microwave-assisted on-chip tryptic digestion method was developed using a domestic microwave, completing the on-chip proteolysis reaction in minutes, whereas the previous on-chip digestion methods by incubation took hours of incubation time. For the model protein chips, antibody-presenting surfaces were prepared, where anti-α-tubulin1 and antibovine serum albumin (BSA) were immobilized on self-assembled monolayers. The resulting digestion efficiency, displaying sequence coverages of 30 and 14% for α-tubulin1 and BSA, respectively, was comparable to the previous time-consuming incubation studies. It allowed the characterization of immunosensed proteins by MASCOT search using peptide mass fingerprinting. In an example of this method for protein chip applications, BSA naturally involved in fetal bovine serum was unambiguously identified on a model protein chip by imaging mass spectrometry. This work shows that biomass spectrometry techniques can be implemented for surface mass spectrometry and biochip applications. Along with recent advances in imaging mass spectrometry, this technique will provide a new opportunity for high-speed, and thus high-throughput in the future, label-free mass spectrometric assays using protein arrays.     

Assessment of microwave-assisted enzymatic digestion by measuring glycated hemoglobin A1c by mass spectrometry

Enzymatic digestion of proteins and analysis of the resulting peptides by mass spectrometry is an established approach in proteomics and in clinical and environmental chemistry. The long digestion times of several hours prevent the fast turnover of samples and results. Qualitative applications showed that microwave radiation profoundly shortens enzymatic digestion. However, its usefulness for quantitative applications had not been assessed. In this study, the microwave-assisted enzymatic digestion of hemoglobin at different temperatures, buffer concentrations, and digestion times was assessed and compared with conventional digestion for the proteolytic enzymes trypsin and Glu-C. A microwave-assisted enzymatic digestion method optimized for digestion time and temperature was applied for the analysis of glycated hemoglobin HbA1c and compared with a reference method. Using trypsin, complete digestion was obtained at 50 degrees C within 20 min. Under these conditions, the digestion efficiency was 20% higher than with conventional trypsin digestion. These effects were not observed with Glu-C as enzyme, probably because of the decreased stability of Glu-C at elevated temperatures in comparison with the trypsin used. The comparison of the optimized microwave-assisted digestion method using trypsin with the reference method for HbA1c using Glu-C gave a close correlation in the results (R2: 0.996). A significant bias of 0.33% HbA1c was observed, with higher values obtained with the microwave-assisted tryptic digest; this finding might have resulted from the use of a different enzyme. This study showed that microwave-assisted enzymatic digestion can substantially reduce digestion times to minutes and can be used in qualitative as well as quantitative applications.     

Antigenicity of heat-treated and trypsin-digested milk samples studied by an optical immunochip biosensor

Abstract  

Individuals with known hypersensitivity or food allergy need to avoid ingestion of provoking food. Correct labelling of allergenic content in manufactured food products and the reliable determination of its residual immunoreactivity after several processing steps are therefore a major concern for the food industry. We evaluated the applicability of a new immunochip biosensor system to reveal the allergenic profile of the whey protein β-lactoglobulin (β-LG) in its natural biological cow’s milk matrix upon processing by tryptic digestion and extensive heat treatment. Colorimetric immunochemical signals generated by gold nanoparticles (Au NPs), in particular their functional optical property based on resonance-enhanced absorption of mirror-reflected light, were directly visible to the ‘naked’ eye of the analyst without the need of any instrumentation or enzyme-substrate for read-out. By using affinity-purified polyclonal rabbit IgG against the native protein, no antigenicity was detected for tryptic fragments. Both heat-denatured whey proteins and cow’s whole milk, however, did not lose their antibody-binding capacity even after a processing time of 20 min at 95°C for the whey proteins, and 60 min at 90°C for the milk, though the immunochemical response was considerably low compared to the unprocessed β-LG. Additionally, cross-reactivity and the false positive as well as false negative predictive value of the chip system were highlighted critically.     

A microchip-based proteolytic digestion system driven by electroosmotic pumping

Microchip-based proteomic analysis requires proteolytic digestion of proteins in microdevices. Enzyme reactors in microdevices, fabricated in glass, silicon, and PDMS substrates, have recently been demonstrated for model protein digestions. The common approach used for these enzyme reactors is employment of a syringe pump(s) to generate hydrodynamic flow, driving the proteins through the reactors. Here we present a novel approach, using electroosmotic flow (EOF) to electrokinetically pump proteins through a proteolytic system. The existence of EOF in the proteolytic system packed with immobilized trypsin gel beads was proven by imaging the movement of a neutral fluorescent marker. Digestions of proteins were subsequently carried out for 12 min, and the tryptic peptides were analyzed independently using capillary electrophoresis (CE) and MALDI-TOF mass spectrometry (MS). The results from CE analysis of the tryptic peptides from the EOF-driven proteolytic system and a conventional water bath digestion were comparable. MALDI-TOF MS was used to identify the parent protein and the tryptic peptides using MS-Fit database searching. The potential utility of the EOF-driven proteolytic system was demonstrated by direct electro-elution of proteins from an acrylamide gel into the proteolytic system, with elution and tryptic digestion achieved in a single step. The EOF-driven proteolytic system, thus, provides a simple way to integrate protein digestion into an electrophoretic micro total analysis system for protein analysis and characterization.     

Accelerated tryptic digestion for the analysis of biopharmaceutical monoclonal antibodies in plasma by liquid chromatography with tandem mass spectrometric detection

Accelerated tryptic digestion of a therapeutic protein including microwave irradiation and thermal transfer by convection at 60 °C and 37 °C was investigated. An analytical setup was devised to follow the protein digestion rate using 1D gel electrophoresis and liquid chromatography coupled a triple quadrupole linear ion trap mass spectrometer. The formation kinetic of its tryptic peptides was monitored in the selected monitoring mode (LC-SRM/MS). Different digestion end points (e.g. 2, 5, 10, 15, 30 and 60 min) as well as an overnight digestion were tested using a therapeutic human monoclonal antibody (mAb) with the goal of its LC-SRM/MS quantification in human plasma. The peptides from the human mAb were generated at different rates and were classified into three categories: (1) the fast forming peptides, (2) the slow forming peptides and (3) the peptides degrading over time. For many monitored peptides, a heating temperature of 37 °C with a 750 rpm mixing applied for at least 30 min provided equivalent results to microwave-assisted digestion and generally allowed the achievement of an equivalent peptide concentration as an overnight digestion carried out at 37 °C. The disappearance of the protein of the heavy and light chains can be monitored by 1D gel electrophoresis but was found not to be representative of the final tryptic peptide concentrations. For quantitative purposes a stable isotope labeled version (¹³C₄, ¹⁵N₁) of the therapeutic protein was used. The labeled protein as internal standard was found to be very efficient to compensate for incomplete digestion or losses during sample preparation.     

Antigenicity of heat-treated and trypsin-digested milk samples studied by an optical immunochip biosensor

Abstract  Individuals with known hypersensitivity or food allergy need to avoid ingestion of provoking food. Correct labelling of allergenic content in manufactured food products and the reliable determination of its residual immunoreactivity after several processing steps are therefore a major concern for the food industry. We evaluated the applicability of a new immunochip biosensor system to reveal the allergenic profile of the whey protein β-lactoglobulin (β-LG) in its natural biological cow’s milk matrix upon processing by tryptic digestion and extensive heat treatment. Colorimetric immunochemical signals generated by gold nanoparticles (Au NPs), in particular their functional optical property based on resonance-enhanced absorption of mirror-reflected light, were directly visible to the ‘naked’ eye of the analyst without the need of any instrumentation or enzyme-substrate for read-out. By using affinity-purified polyclonal rabbit IgG against the native protein, no antigenicity was detected for tryptic fragments. Both heat-denatured whey proteins and cow’s whole milk, however, did not lose their antibody-binding capacity even after a processing time of 20 min at 95°C for the whey proteins, and 60 min at 90°C for the milk, though the immunochemical response was considerably low compared to the unprocessed β-LG. Additionally, cross-reactivity and the false positive as well as false negative predictive value of the chip system were highlighted critically. Graphical abstract        

Trypsin and MALDI matrix pre‐coated targets simplify sample preparation for mapping proteomic distributions within biological tissues by imaging mass spectrometry

Prefabricated surfaces containing α‐cyano‐4‐hydroxycinnamic acid and trypsin have been developed to facilitate enzymatic digestion of endogenous tissue proteins prior to matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). Tissue sections are placed onto slides that were previously coated with α‐cyano‐4‐hydroxycinnamic acid and trypsin. After incubation to promote enzymatic digestion, the tissue is analyzed by MALDI IMS to determine the spatial distribution of the tryptic fragments. The peptides detected in the MALDI IMS dataset were identified by Liquid chromatography‐tandem mass spectrometry/mass spectrometry. Protein identification was further confirmed by correlating the localization of unique tryptic fragments originating from common parent proteins. Using this procedure, proteins with molecular weights as large as 300 kDa were identified and their distributions were imaged in sections of rat brain. In particular, large proteins such as myristoylated alanine‐rich C‐kinase substrate (29.8 kDa) and spectrin alpha chain, non‐erythrocytic 1 (284 kDa) were detected that are not observed without trypsin. The pre‐coated targets simplify workflow and increase sample throughput by decreasing the sample preparation time. Further, the approach allows imaging at higher spatial resolution compared with robotic spotters that apply one drop at a time. Copyright © 2016 John Wiley & Sons, Ltd.     

Quick quantification of proteins by MALDI

Previously, we reported that the matrix‐assisted laser desorption ionization spectrum of a peptide became reproducible when an effective temperature was held constant. Using a calibration curve drawn by plotting the peptide‐to‐matrix ion abundance ratio versus the peptide concentration in a solid sample, a peptide could be quantified without the use of any internal standard. In this work, we quantified proteins by quantifying their tryptic peptides with the aforementioned method. We modified the digestion process; e.g. disulfide bonds were not cleaved, so that hardly any reagent other than trypsin remained after the digestion process. This allowed the preparation of a sample by the direct mixing of a digestion mixture with a matrix solution. We also observed that the efficiency of the matrix‐to‐peptide proton transfer, as measured by its reaction quotient, was similar for peptides with arginine at the C‐terminus. With the reaction quotient averaged over many such peptides, we could rapidly quantify proteins. Most importantly, no peptide standard, not to mention its isotopically labeled analog, was needed in this method. Copyright © 2015 John Wiley & Sons, Ltd.     

On-Line multitasking analytical proteomics: how to separate, reduce, alkylate and digest whole proteins in an on-Line multidimensional chromatography system coupled to MS

An on-Line multidimensional system has been developed, consisting of pH gradient strong anion exchange chromatography of native proteins in the first dimension with subsequent trapping and on-column reduction/alkylation on C4 trap columns and RP separation of the alkylated proteins in the second dimension followed by on-column tryptic digestion and electrospray MS detection. The system was evaluated using model proteins and a human urine sample. Compared to the commonly used in-solution alkylation method, the developed on-column method provides an equivalent efficiency. The recovery from the C4 trap columns of the alkylated proteins relative to the native state was from 94 to 102%. On-column tryptic digestion was satisfactory for many, but not for all proteins. The whole analytical procedure was performed on-Line with packed capillary columns for a total time of 320 min for the first ion exchange fraction, with additional 60 min for each subsequent fraction.