[Ion chromatography-pulsed amperometry method for determination of trace hydrogen sulfide in air].

Hydrogen sulfide (H2S) is a pervasive gaseous pollutant that emits the characteristic odor of rotten gas, even at low concentrations. It is generated during various industrial processes, including petroleum and natural gas refining, mining operations, wastewater treatment activities, and refuse disposal practices. According to statistics from the World Health Organization (WHO), over 70 occupations are exposed to H2S, rendering it a key monitoring factor in occupational disease detection. Although H2S has legitimate uses in the chemical, medical, and other fields, prolonged exposure to this gas can cause severe damage to the respiratory and central nervous systems, as well as other organs in the human body. Moreover, the substantial release of H2S into the environment can lead to significant pollution. This noxious substance has the potential to impair soil, water, and air quality, while disrupting the equilibrium of the surrounding ecosystems. Therefore, sulfide has become one of the most commonly measured substances for environmental monitoring worldwide. Achieving the stable enrichment and accurate detection of low-level H2S is of great significance. Common methods for detecting this gas include spectrophotometry, chemical analysis, gas chromatography, rapid field detection, and ion chromatography. Although these methods provide relatively reliable results, they suffer from limitations such as high detection cost, low recovery, lack of environmental friendliness, and imprecise quantification of low-concentration H2S. Furthermore, the sampling processes involved in these methods are complex and require specialized equipment and electrical devices. Additionally, approximately 20% of the sulfides in a sample are lost after 2 h in a conventional alkaline sodium hydroxide solution, causing difficulties in preservation and detection. In this study, an accurate, efficient, and cost-saving method based on ion chromatography-pulse amperometry was developed for H2S determination. A conventional IonPac AS7 (250 mm×4 mm) anion-exchange column was employed, and a new eluent based on sodium hydroxide and sodium oxalate was used to replace the original sodium hydroxide-sodium acetate eluent. The main factors influencing the separation and detection performance of the proposed method, including the pulse amperage detection potential parameters and integration time, as well as the type and content of additives in the stabilizing solution, were optimized. The results showed that the proposed method had a good linear relationship between 10 and 3000 µg/L, with correlation coefficients (r2) of up to 0.999. The limits of detection (S/N=3) and quantification (S/N=10) were 1.53 and 5.10 µg/L, respectively. The relative standard deviations (RSDs) of the peak area and retention time of sulfides were less than 0.2% (n=6). The new method exhibited excellent stability, with up to 90% reduction in reagent costs. Compared with conventional ion chromatography-pulse amperometry, this method is more suitable for detecting low concentrations of sulfides in actual samples. Sulfides in a 250 mmol/L sodium hydroxide-0.8% (mass fraction) ethylenediaminetetraacetic acid disodium salt solution were effectively maintained for over 10 h. The new stabilizer significantly improved the reliability of both large-scale and long-term detection. The recovery of the proposed method was investigated by combining the system with a badge-type passive sampler. This sampling method requires no power devices; it is inexpensive, simple to operate, and can realize long-term sampling without the need for skilled personnel. Moreover, it can overcome the influence of short-term changes in pollutant concentration. The sampling results have high reference value for large-scale intervention-less pollutant monitoring in ultraclean rooms, museum counters, and other places. The results demonstrated that the recovery of the proposed method was greater than 95% for the blank sample and 80% for the sample plus standard solution. Finally, the newly established method was applied to determine H2S levels in air samples collected via passive sampling at school garbage stations. The measured results did not exceed the national limit.

[Application of ion chromatographic fingerprint analysis for quality evaluation of tobacco flavors].

Tobacco flavor, an important tobacco additive, is an essential raw material in cigarette production that can effectively improve the quality of tobacco products, add aroma and taste, and increase the suction flavor. The quality consistency of tobacco flavors affects the quality stability of branded cigarettes. Therefore, the quality control of tobacco flavors is a major concern for cigarette and flavor manufacturers. Physical and chemical indices, odor similarity, and sensory efficacy are employed to evaluate the quality of tobacco flavors, and the analysis of chemical components in tobacco flavors is usually conducted using gas chromatography (GC) and high performance liquid chromatography (HPLC). However, because the composition of tobacco flavors is complex, their quality cannot be fully reflected using a single component or combination of components. Therefore, establishing an objective analytical method for the quality control of tobacco flavors is of extreme importance. Chromatographic fingerprint analysis is routinely used for the discriminative analysis of tobacco flavors. Chromatographic fingerprints refer to the general characteristics of the concentration profiles of different chemical compounds. In the daily procurement process, fingerprints established by GC and HPLC are effective for the evaluation and identification of tobacco flavors. However, given continuous improvements in aroma-imitation technology, some flavors with high similarity cannot be directly distinguished using existing methods. In this study, a method for the determination of organic acids and inorganic anions in tobacco flavors based on ion chromatography (IC) was developed to ensure the quality consistency of tobacco flavors. A 1.0 g sample of tobacco flavors and 10 mL of deionized water were mixed and vibrated for 30 min. The aqueous sample solution was passed through a 0.45 µm membrane filter and RP pretreatment column in succession to eliminate interferences and then subjected to IC. Standard solutions containing nine organic acids and seven inorganic anions were used to identify the anions in the tobacco flavors, and satisfactory reproducibility was obtained. The relative standard deviations (RSDs) for retention times and peak areas were <0.71% and <6.02%, respectively. The chromatographic fingerprints of four types of tobacco flavors (samples A-D) from five different batches were obtained. Nine tobacco flavor samples from different manufacturers (samples AY1-AY3, BY1-BY2, CY1-CY2, DY1-DY2) were also analyzed to obtain their chromatographic fingerprints. Hierarchical cluster and similarity analyses were used to evaluate the quality of tobacco flavors from different manufacturers. Hierarchical clustering refers to the process of subdividing a group of samples into clusters that exhibit a high degree of intracluster similarity and intercluster dissimilarity. The dendrograms obtained using SPSS 12.0 indicated good quality consistency among the samples in different batches. Samples AY3, BY2, CY2, and DY1 clustered with the batches of standard tobacco flavors. Therefore, hierarchical cluster analysis can effectively distinguish the quality of products from different manufacturers. The Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (version 2.0) was used to evaluate the similarity between the standard tobacco flavors and products from different manufacturers. Among the samples analyzed, samples AY3, BY2, CY2, and DY1 showed the highest similarity values (>97.7%), which was consistent with the results of the hierarchical cluster analysis. This finding indicates that IC combined with chromatographic fingerprint analysis could accurately determine the quality of tobacco flavors. GC combined with ultrasonic-assisted liquid-liquid extraction was also used to analyze the tobacco flavors and verify the accuracy of the proposed method. Compared with GC coupled with ultrasonic-assisted liquid-liquid extraction, IC demonstrated more significant quality differences among certain tobacco flavors.

Role of Charge Heterogeneity on Physical Stability of Monoclonal Antibody Biotherapeutic Products.

PURPOSE: Chemical modifications in monoclonal antibodies can change hydrophobicity, charge heterogeneity as well as conformation, which eventually can impact their physical stability. In this study, the effect of the individual charge variants on physical stability and aggregation propensity in two different buffer conditions used during downstream purification was investigated. METHODS: The charge variants were separated using semi-preparative cation exchange chromatography and buffer exchanged in the two buffers with pH 6.0 and 3.8. Subsequently each variant was analysed for size heterogeneity using size exclusion chromatography and dynamic light scattering, conformational stability, colloidal stability, and aggregation behaviour under accelerated stability conditions. RESULTS: Size variants in each charge variant were similar in both pH conditions when analyzed without extended storage. However, conformational stability was lower at pH 3.8 than pH 6.0. All charge variants showed similar apparent melting temperature at pH 6.0. In contrast, at pH 3.8 variants A3, A5, B2, B3 and B4 display lower Tm, suggesting reduced conformational stability. Further, A2, A3 and A5 exhibit reduced colloidal stability at pH 3.8. In general, acidic variants are more prone to aggregation than basic variants. CONCLUSION: Typical industry practice today is to examine in-process intermediate stability with acidic species and basic species taken as a single category each. We suggest that perhaps stability evaluation needs to be performed at specie level as different acidic or basic species have different stability and this knowledge can be used for clever designing of the downstream process to achieve a stable product.

Anion Exchange Chromatography-Mass Spectrometry to Characterize Proteoforms of Alpha-1-Acid Glycoprotein during and after Pregnancy.

Alpha-1-acid glycoprotein (AGP) is a heterogeneous glycoprotein fulfilling key roles in many biological processes, including transport of drugs and hormones and modulation of inflammatory and immune responses. The glycoform profile of AGP is known to change depending on (patho)physiological states such as inflammatory diseases or pregnancy. Besides complexity originating from five N-glycosylation sites, the heterogeneity of the AGP further expands to genetic variants. To allow in-depth characterization of this intriguing protein, we developed a method using anion exchange chromatography (AEX) coupled to mass spectrometry (MS) revealing the presence of over 400 proteoforms differing in their glycosylation or genetic variants. More precisely, we could determine that AGP mainly consists of highly sialylated higher antennary structures with on average 16 sialic acids and 0 or 1 fucose per protein. Interestingly, a slightly higher level of fucosylation was observed for AGP1 variants compared to that of AGP2. Proteoform assignment was supported by integrating data from complementary MS-based approaches, including AEX-MS of an exoglycosidase-treated sample and glycopeptide analysis after tryptic digestion. The developed analytical method was applied to characterize AGP from plasma of women during and after pregnancy, revealing differences in glycosylation profiles, specifically in the number of antennae, HexHexNAc units, and sialic acids.

Highly sensitive two-dimensional ion chromatography mass spectrometry method for nitrite determination in hydroxypropyl methylcellulose.

Due to their potential adverse health effects, some N-nitrosamines in drug products are strictly regulated with very low maximum daily intake limits. Nitrosamines can be formed from the reaction of nitrite and secondary or tertiary amines when both species co-exist in the drug synthesis or formulation process. One key strategy to mitigate nitrosamine risk in drugs is to select low-nitrite containing pharma excipients for formulation. It is necessary to develop a sensitive method for trace nitrite determination in pharma excipients as it enables drug producers to study nitrosamine formation kinetics and select excipient suppliers. This study details the development and validation of a two-dimensional ion chromatography mass spectrometry (2D-IC/MS) method for trace nitrite determination in hydroxypropyl methylcellulose (HPMC), one of the most important pharmaceutical excipients used in many drug formulations. The 2D-IC system was operated in heart-cutting mode with a concentrator column coupling the two dimensions. A standard bore anion-exchange column was used in the first dimension (1D) to enable a large volume injection for increased sensitivity and provide improved resolution between nitrite and the interfering chloride peak. A high efficiency microbore anion-exchange column with different selectivity was used in the second dimension (2D) to resolve nitrite from other interfering species. The use of 2D-IC resulted in significantly improved resolution, solving the sensitivity loss issue due to ion suppression from an otherwise 1D separation. MS detection with selective ion monitoring and isotope labeled nitrite internal standard further improve the method specificity, accuracy, and ruggedness, as compared with conductivity detection. For trace determination, it is also extremely important to have a clean blank. For this purpose, a novel cleaning procedure using a strong anion wash was developed to remove nitrite contamination from labware. The optimized method was validated with linearity of nitrite in the concentration range of 18.5-5005.8 ng/g having a regression coefficient of >0.9999, precision with RSD at 3.5-10.1 % and recovery of 90.5-102.4 %. The limit of detection and limit of quantitation were 8.9 and 29.6 ng/g relative to the HPMC sample, or equivalent to 89 and 296 pg/g in the sample solution, respectively.

Analysis of Polypeptides by Amino Acid Analysis.

Amino acid analysis is an accurate method for the composition and quantitation of polypeptides and among these synthetic peptides. Combined with mass spectrometry, it yields a reliable control of peptide quality and quantity prior to conjugation and immunization.Initially peptides are hydrolyzed, preferably in the gas phase, with 6-M HCl at 110 °C for 20-24 h and the resulting amino acids analyzed by chromatography, where the most reliable form is ion exchange chromatography with post-column ninhydrin derivatization. Depending on the hydrolysis conditions, tryptophan is destroyed, and likewise cysteine, unless derivatized, and the amides, glutamine, and asparagine are deamidated to glutamic acid and aspartic acid, respectively. Three different ways of calculating results are suggested, and taking the above limitations into account, a quantitation better than 5% can usually be obtained.

Ion Chromatography and Related Techniques in Carbohydrate Analysis: A Review.

Ion chromatography and related techniques have been the most popular separation methods used in the determination of organic and inorganic anions and cations, predominantly in water and wastewater samples. Making progress in their development and introducing new stationary phases, methods of detection and preparation of samples for analyses have given rise to the broadening of their analytical range. Nowadays, they are also used for substances that are not ionic by nature but can convert to such forms under certain conditions. These encompass, among others, carbohydrates, whose role and significance in humans' lives and environment is invaluable. Their presence in the air is mostly due to the industrial burning of biomass for energy production purposes. In addition, the content of sugars in plants, fruits and vegetables, constituting the base of human diets, affects our health condition. Given that, there is not only a need for their determination by means of routine methods but also for searching for novel analytical solutions. Based on literature data from the past decade, this paper presents the possibilities and examples of applications regarding ion chromatography and related techniques for the determination of carbohydrates in environmental samples, biomass and plants constituting food or raw materials for food production. Attention has been paid to the virtues and limitations of the discussed separation methods in this respect. Moreover, perspectives on their development have been defined.

Structural study of a light chain mispaired bispecific predicts mechanism of downstream separation.

Bispecific antibodies expressed and assembled from a single upstream culture require the correct balance and pairing of four different heavy and light chains (HC and LC). The increased potential for chain-mispaired species challenges the downstream purification of this new format. While clearance of HC-mispaired species, including homodimers and half-antibodies, has been assessed, removal of LC mispairs requires a more stringent approach. Here, we report two case studies in which separation is achieved, as well as the structural basis of these separations: (A) In the first case, a main species with a positively charged patch in the correctly formed variable fragment (Fv) is disrupted when paired with the wrong LC. This LC-mispaired variant binds more weakly to a cation exchange resin and can be washed off in a chromatography step. (B) A second molecule whose LC mispair introduces a negative-charge patch and hydrophobic patch in close proximity, presenting increased binding to a multimodal anion exchange resin. This LC-mispaired variant can be retained on the column under conditions in which the bispecific is recovered. In both case studies, the molecular structural analysis by protein surface properties models correlated well with the chromatography experiments. The comprehensive interpretation of experimental and computational results has provided a better understanding of strategies and potential applications for predicting the downstream purification of complex molecules.

Pretreatment methods in ion chromatography: A review.

As an advanced analytical technology, Ion Chromatography (IC) has been widely used in various fields. At present, it is faced with the challenges of sample complexity and instrument precision. It is necessary to select appropriate pretreatment methods to achieve sample preparation and protect the instruments. Therefore, this paper reviews several commonly used sample pretreatment technologies in IC, focusing on sample digestion and purification techniques. Additionally, we introduce some advanced IC technologies and automatic sample processing devices. We provide a comprehensive summary of the basic principles, primary applications and the advantages and disadvantages of each method. Pretreatment methods should be carefully selected and optimized on the specific characteristics of the sample and the ions to be measured, in order to achieve better analysis results.

Competitive protein adsorption in isocratic anion exchange chromatography.

Protein chromatography is the dominant method of purification of biopharmaceuticals. Although all practical chromatography involves competitive absorption and separation of M. species, competitive protein absorption has remained inadequately understood. We previously introduced the measurement of equilibrium protein adsorption isotherms with all intensive variables held constant, including competitor concentration. In this work, we introduce isocratic chromatographic retention measurements of dynamic protein adsorption in the presence of a constant concentration of a competitor protein. These measurements are achieved by establishing a dynamic equilibrium with a constant concentration of competitor (insulin) in the mobile phase flowing through an ion exchange adsorbent column and following the behavior of a test protein (α-lactalbumin) injected into this environment. We observed decreased retention times for α-lactalbumin in presence of the competitor. The presence of competitor also reduces the heterogeneity of the sites available for adsorption of the test protein. This investigation provides an approach to fundamental understanding of competitive dynamics of multicomponent protein chromatography.

Purification Process for a Secreted Protein Produced in Insect Cells.

The Baculovirus Expression Vector System (BEVS) is used with cultured insect cells to produce a wide variety of heterologous proteins, which can be secreted into the culture medium during the transient infection process (Smith et al. Mol Cell Biol 12:2156-2165, 1983). When the infection process is complete, centrifugation is often used to separate the desired protein from the spent insect cells. The desired product in the harvested supernatant is contaminated with baculovirus, amino acids, lipids, detergents, oils, lysed cells from the infection process, genomic DNA from the insect cells, and proteases due to the lytic nature of the baculovirus infection process and many other contaminants (Ikonomou et al. Appl Microbiol Biotechnol 62:1-20, 2003). All these contaminants that are present in the centrifuged supernatant with the desired secreted protein make the initial chromatographic capture step critical for effective purification of the desired protein. A purification scheme will be outlined for a slightly acidic secreted protein using cation exchange chromatography (Lundanes et al. Chromatography: basic principles, sample preparations and related methods, 1st edn. Wiley, 2013).

One-Step Purification Strategy for Cowpea Chlorotic Mottle Virus-Like Particles Produced by the IC-BEVS.

Virus-like particles (VLP) of the cowpea chlorotic mottle virus (CCMV), a plant virus, have been shown to be safe and noncytotoxic vehicles for delivering various cargos, including nucleic acids and peptides, and as scaffolds for presenting epitopes. Thus, CCMV-VLP have acquired increasing attention to be used in fields such as gene therapy, drug delivery, and vaccine development. Regardless of their production method, most reports purify CCMV-VLP through a series of ultracentrifugation steps using sucrose density gradient ultracentrifugation, which is a complex and time-consuming process. Here, the use of anion exchange chromatography is described as a one-step protocol for purification of CCMV-VLP produced by the insect cell-baculovirus expression vector system (IC-BEVS).

Integrated Platform for Large-Scale Quantitative Profiling of Phosphotyrosine Signaling Complexes Based on Cofractionation/Mass Spectrometry and Complex-Centric Algorithm.

The scarcity and dynamic nature of phosphotyrosine (pTyr)-modified proteins pose a challenge for researching protein complexes with pTyr modification, which are assembled through multiple protein-protein interactions. We developed an integrated complex-centric platform for large-scale quantitative profiling of pTyr signaling complexes based on cofractionation/mass spectrometry (CoFrac-MS) and a complex-centric algorithm. We initially constructed a trifunctional probe based on pTyr superbinder (SH2-S) for specifically binding and isolation of intact pTyr protein complexes. Then, the CoFrac-MS strategy was employed for the identification of pTyr protein complexes by integrating ion exchange chromatography in conjunction with data independent acquisition mass spectrometry. Furthermore, we developed a novel complex-centric algorithm for quantifying protein complexes based on the protein complex elution curve. Utilizing this algorithm, we effectively quantified 216 putative protein complexes. We further screened 21 regulated pTyr protein complexes related to the epidermal growth factor signal. Our study engenders a comprehensive framework for the intricate examination of pTyr protein complexes and presents, for the foremost occasion, a quantitative landscape delineating the composition of pTyr protein complexes in HeLa cells.

[Recent progress of chromatographic techniques for antibody purification].

Antibody drugs are becoming increasingly popular in disease diagnosis, targeted therapy, and immunoprevention owing to their characteristics of high targeting ability, strong specificity, low toxicity, and mild side effects. The demand for antibody drugs is steadily increasing, and their production scale is expanding. Upstream cell culture technology has been greatly improved by the high-capacity production of monoclonal antibodies. However, the downstream purification of antibodies presents a bottleneck in the production process. Moreover, the purification cost of antibodies is extremely high, accounting for approximately 50%-80% of the total cost of antibody production. Chromatographic technology, given its selectivity and high separation efficiency, is the main method for antibody purification. This process usually involves three stages: antibody capture, intermediate purification, and polishing. Different chromatographic techniques, such as affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, and temperature-responsive chromatography, are used in each stage. Affinity chromatography, mainly protein A affinity chromatography, is applied for the selective capture and purification of antibodies from raw biofluids or harvested cell culture supernatants. Other chromatographic techniques, such as ion-exchange chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography, are used for intermediate purification and antibody polishing. Affinity biomimetic chromatography and hydrophobic charge-induction chromatography can produce antibodies with purities comparable with those obtained through protein A chromatography, by employing artificial chemical/short peptide ligands with good selectivity, high stability, and low cost. Temperature-responsive chromatography is a promising technique for the separation and purification of antibodies. In this technique, antibody capture and elution is controlled by simply adjusting the column temperature, which greatly eliminates the risk of antibody aggregation and inactivation under acidic elution conditions. The combination of different chromatographic methods to improve separation selectivity and achieve effective elution under mild conditions is another useful strategy to enhance the yield and quality of antibodies. This review provides an overview of recent advances in the field of antibody purification using chromatography and discusses future developments in this technology.

Optimization of High-Activity earthworm fibrinolytic enzyme extraction methods and protein component analysis.

OBJECTIVE: Optimize the extraction process of earthworm fibrinolytic enzyme. METHODS: Chinese common earthworms underwent a series of purification processes, including grinding, salting out, hydrophobic medium chromatography, ammonium sulfate precipitation, and ion exchange chromatography, to obtain purified earthworm fibrinolytic enzyme. RESULTS: Utilizing Pheretima aspergillum as the starting material, we discovered that the specific activity of lumbrokinase extracted via ammonium sulfate precipitation was 58 U/mg, noticeably surpassing that achieved through heat precipitation and ethanol precipitation methods. After undergoing two rounds of chromatographic separations employing hydrophobic affinity chromatography and anion exchange chromatography, the specific activity of the lumbrokinase protein soared to 9267 U/mg, significantly exceeding the 3,178 U/mg specific activity attained through industrial extraction methods. DISCUSSION: The development of a novel crude extraction method for lumbrokinase protein can significantly boost its activity and purity. The discovery of a high-efficiency purification method and the identification of protein components within highly active lumbrokinase pave the way for further investigations into these proteins.

Separation of full, empty, and partial adeno-associated virus capsids via anion-exchange chromatography with continuous recycling and accumulation.

The field of recombinant adeno-associated virus (rAAV) gene therapy has attracted increasing attention over decades. Within the ongoing challenges of rAAV manufacturing, the co-production of impurities, such as empty and partial capsids containing no or truncated transgenes, poses a significant challenge. Due to their potential impact on drug efficacy and clinical safety, it is imperative to conduct comprehensive monitoring and characterization of these impurities prior to the release of the final gene therapy product. Nevertheless, existing analytical techniques encounter notable limitations, encompassing low throughput, long turnaround times, high sample consumption, and/or complicated data analysis. Chromatography-based analytical methods are recognized for their current Good Manufacturing Practice (cGMP) alignment, high repeatability, reproducibility, low limit of detection, and rapid turnaround times. Despite these advantages, current anion exchange high pressure liquid chromatography (AEX-HPLC) methods struggle with baseline separation of partial capsids from full and empty capsids, resulting in inaccurate full-to-empty capsid ratio, as partial capsids are obscured within peaks corresponding to empty and full capsids. In this study, we present a unique analytical AEX method designed to characterize not only empty and full capsids but also partial capsids. This method utilizes continuous N-Rich chromatography with recycling between two identical AEX columns for the accumulation and isolation of partial capsids. The development process is comprehensively discussed, covering the preparation of reference materials representing full (rAAV-LacZ), partial (rAAV-GFP), and empty (rAAV-empty) capsids, N-rich method development, fraction analysis, determination of fluorescence response factors between capsid variants, and validation through comparison with other comparative techniques.

Online Mixed-Bed Ion Exchange Chromatography for Native Top-Down Proteomics of Complex Mixtures.

Native top-down mass spectrometry (nTDMS) allows characterization of protein structure and noncovalent interactions with simultaneous sequence mapping and proteoform characterization. The majority of nTDMS studies utilize purified recombinant proteins, with significant challenges hindering application to endogenous systems. To perform native top-down proteomics (nTDP), where endogenous proteins from complex biological systems are analyzed by nTDMS, it is essential to separate proteins under nondenaturing conditions. However, it remains difficult to achieve high resolution with MS-compatible online chromatography while preserving protein tertiary structure and noncovalent interactions. Herein, we report the use of online mixed-bed ion exchange chromatography (IEC) to enable separation of endogenous proteins from complex mixtures under nondenaturing conditions, preserving noncovalent interactions for nTDP analysis. We have successfully detected large proteins (>146 kDa) and identified endogenous metal-binding and oligomeric protein complexes in human heart tissue lysate. The use of a mixed-bed stationary phase allowed retention and elution of proteins over a wide range of isoelectric points without altering the sample or mobile phase pH. Overall, our method provides a simple online IEC-MS platform that can effectively separate proteins from complex mixtures under nondenaturing conditions and preserve higher-order structure for nTDP applications.

Site-specific identification and quantitation of endogenous SUMOylation based on SUMO-specific protease and strong anion exchange chromatography.

Small ubiquitin-like modifier (SUMO) modification regulates various eukaryotic cellular processes and plays a pivotal role in interferon (IFN)-mediated antiviral defense. While immunoprecipitation enrichment method is widely used for proteome-wide analysis of endogenous SUMOylation, the inability to target all SUMO forms and high cost of antibodies limited its further application. Herein, we proposed an antibody-free enrichment method based on SUMO-specific protease and strong anion exchange chromatography (SPAX) to globally profile the endogenous SUMOylation. The SUMO1/2/3-modified peptides could be simultaneously enriched by SAX chromatography by utilizing its electrostatic interaction with SUMO1/2/3 remnants, which contained multiple aspartic acids (D) and glutamic acids (E). To remove the co-enriched D/E-containing peptides which might interfere with the detection of low-abundance SUMOylated peptides, SUMO-specific protease was used to cleave the SUMO1/2/3 remnants from enriched SUMOylated peptides. As the deSUMOylated peptides lost SUMO remnants, their interaction with SAX materials became weaker, and the D/E-containing peptides could thus be depleted through the second SAX separation. The SPAX method identified over twice the SUMOylated sites than using SAX method only, greatly improving the identification coverage of endogenous SUMOylated sites. Our strategy was then applied to the site-specific identification and quantification of endogenous SUMOylation in A549 cells stimulated by IFN-γ for the first time. A total of 226 SUMOylated sites on 146 proteins were confidently identified, among which multiple up-regulated sites were involved in IFN-mediated antiviral defense, demonstrating the great promise of SPAX to globally profile and discover endogenous SUMOylation with significant biological functions.

Multiple, simultaneous, independent gradients for a versatile multidimensional liquid chromatography. Part II: Application 3 - Scouting optimization strategies for separation of monoclonal antibodies by dual simultaneous independent gradients of pH & salt on a weak cation exchange stationary phase.

In previous publications we have described the pISep dual simultaneous, independent gradients (DSIGs) liquid chromatography (LC) for uncoupling gradients of non-buffering solute (NaCl, urea or acetonitrile) from externally generated pH gradients. In DSIGs the shape and slope of the [salute] gradient does not depend on the shape and slope of the pH gradient. The technique allows in a single run true simultaneous two dimensional LC separation of complex protein mixtures on various stationary phases including anion, cation exchangers (AEX, CEX), reversed phase (RP), mixed mode and mixed bed. Using a humanized IgG1 (HIgG1) monoclonal antibody (MAb) and a variety of pH & [NaCl] DSIGs, we show that most of MAb isoforms can be successfully separated from each other. These experimental observations are supported by an initial theoretical argument presented here predicting an overall improvement of all MAb isoforms separation by DSIGs of pH & [NaCl]. Theoretical calculations predict that, in general, there exists an optimal non-zero isocratic salt concentration in a pH gradient separation that will resolve isoforms close in binding energy, but a wide range of salt concentrations will be required for acceptable resolution of all isoforms. Theory also predicts better separation of weaker rather than stronger binding isoforms. Experimentally, we have found that no one set of DSIGs LC conditions could optimally baseline resolve all identifiable MAb isoforms in a single run of reasonable duration. The versatility and simplicity of the pH & [NaCl] pISep DSIGs LC allows fast, automated scouting of protein separations over any range of pH from 2.4 to 10.8 and [NaCl] from 0 to 1 M without changing the chemistry of the buffering system. Due to the universal applicability of the pISep buffering system in IEX LC, the researcher is given a powerful tool to easily develop pH & [NaCl] DSIGs protocols that vary mobile phase compositions to achieve high resolution separations of targeted proteins.

Sequential diethylaminoethyl dextran-grafting and diethylaminoethyl modification improve the adsorption performance of anion exchangers.

Ion exchangers with high adsorption capacity, fast mass transfer, and high salt-tolerance synchronously are highly desired for high-performance protein purification. Here, we propose a sequential diethylaminoethyl dextran-grafting and diethylaminoethyl chloride modification strategy to achieve high-performance anion exchangers. The advantages of the double-modification strategy lie in: (1) the introduction of diethylaminoethyl in the second modification has no diffusion limitation due to the small molecular size, thus a high ionic capacity; (2) the grafting ligands not only provide three-dimensional adsorption space for high adsorption capacitybut alsofacilitate surface diffusion of protein by chain delivery. The maximum adsorption capacity of the obtained anion exchangers for bovine serum albumin reaches 333 mg/mL, the ratio of effective pore diffusivity (De) to free solution diffusivity (D0) reaches 0.69, and the adsorption amount reaches 97 mg/mL even in 100 mmol/L NaCl concentration,. All these results demonstrate the proposed sequential modification strategy are promising for the preparation of high-performance ion exchangers.