Strong Cation Exchange Chromatography Research Articles

Comprehensive Mapping of Cyclotides from Viola philippica by Using Mass Spectrometry-Based Strategy.

Cyclotides are plant cyclic peptides with exceptional stability and diverse bioactivity, making them promising candidates for biomedical applications. Therefore, the study of cyclotides has attracted increasing attention in recent years. However, the existing cyclotide detection methods face limitations in sensitivity, accuracy, and reliability. To address these challenges, we developed an integrated strategy using a combination of strong cation exchange chromatography techniques for removing interfering small molecules, Orbitrap Exploris 480 mass spectrometry (OEMS); this is a detection and database searching-based method for cyclotide verification, which greatly improved the sensitivity, accuracy, and reliability of cyclotide identification. This strategy was subsequently employed for cyclotide mapping in Viola with a minute amount of starting tissue, resulting the identification of 65 known and 18 potentially novel cyclotides, which is the largest dataset of cyclotides for Viola philippica. This strategy provided valuable insights into the cyclotide diversity and distribution in V. philippica, with potential applications in drug discovery and other biomedical fields.

One-step N-Terminomics Based on Isolation of Protein N-Terminal Peptides From LysargiNase Digests by Tip-Based Strong Cation Exchange Chromatography

We have developed a one-step isolation method for protein N-terminal peptides from LysargiNase digests by pipette tip-based strong cation exchange (SCX) chromatography. This CHAMP-N (CHromatographic AMplification of Protein N-terminal peptides) method using disposable and parallel-processable SCX tips instead of conventional HPLC SCX columns facilitates simple, sensitive, reproducible, and high-throughput N-terminomic profiling without sacrificing the high identification numbers and selectivity achieved by the HPLC-based method. By applying the CHAMP-N method to HEK293T cells, we identified novel cleavage sites for signal and transit peptides and non-canonical translation initiation sites. Finally, for proteome-wide terminomics, we present a simple and comprehensive N- and C-terminomics platform employing three different tip-based approaches, including CHAMP-N, in which protease digestion and one-step isolation by tip LC are commonly used to achieve complementary terminome coverages.

A combination of multiple LC-MS approaches for the comprehensive characterization of cysteine-linked ADCs

Antibody-drug conjugates (ADCs) represent the forefront of the next generation of biopharmaceuticals. An ADC typically comprises an antibody covalently linked to a cytotoxic drug via a linker, resulting in a highly heterogeneous product. This study focuses on the analysis of a custom-made cysteine-linked ADC. Initially, we developed a LC-MS-based characterization workflow using brentuximab vedotin (Adcetris®), encompassing native intact MS, analysis of reduced chains and subunits under denaturing condition, peptide mapping and online strong cation exchange chromatography coupled with UV and mass spectrometry detection (SCX-UV-MS) applied for brentuximab vedotin first time reported. Subsequently, we applied this in-depth characterization workflow to a custom-made cysteine-linked ADC. The measured drug-to-antibody ratio(DAR) of this ADC is 6.9, further analysis shown that there is a small amount of unexpected over-conjugation. Over-conjugation sites were successfully identified using multiple UHPLC-MS based characterization techniques. Also, one competitively cysteine-conjugated impurity was observed in native intact MS results, by combing native intact MS, reduced chains, subunit analysis and peptide mapping results, the impurity conjugation sites were also identified. Since this molecule is at early development stage, this provides important information for conjugation process improvement and link-drug material purification. SCX-UV-MS approach can separate the custom-made cysteine-linked ADC carrying different payloads and reduce the complexity of the spectra. The integrated approach underscores the significance of combining the SCX-UV-MS online coupling technique with other characterization methods to elucidate the heterogeneity of cysteine-linked ADCs.

Implementation of a LC-MS based multi-attribute method (MAM) and intact multi-attribute method (iMAM) workflow for the characterisation of a GLP-Fc fusion protein

Over the past few years, the implementation of mass spectrometry (MS) in QC laboratories has become a more common occurrence. The multi-attribute method (MAM), and emerging intact multi-attribute method (iMAM), are powerful analytical tools utilising liquid chromatography−mass spectrometry (LC-MS) methods that enable the monitoring of critical quality attributes (CQAs) in biotherapeutic proteins in compliant settings. Both MAM and iMAM are intended to replace or supplement several conventional assays with a single LC-MS method utilising MS data in combination with robust, semi-automated data processing workflows. MAM and iMAM workflows can also be implemented into current Good Manufacturing Practices environments due to the availability of CFR 11 compliant chromatography data system software. In this study, MAM and iMAM are employed for the analysis of 4 batches of a glucagon-like peptide-Fc fusion protein. MAM approach involved a first the discovery phase for the identification of CQAs and second, the target monitoring phase of the selected CQAs in other samples. New peak detection was performed on the data set to determine the appearance, absence or change of any peak. For native iMAM workflow both size exclusion and strong cation exchange chromatography were optimized for the identification and monitoring of CQAs at the intact level.

First report of antioxidant potential of peptide fraction derived from Colaconema formosanum (Rhodophyta) protein hydrolysates

Foods that contain natural antioxidants can improve people's health and performance and make them more resistant to environmental stressors like diseases, lousy living conditions, and heat stress. Marine algae are well known for their function as an antioxidant. Colaconema formosanum is a new rhodophyte discovered in Taiwan and its bioactive activities are rarely studied. This work aims to prepare the protein hydrolysate fraction with antioxidant properties in C. formosanum using single enzyme or complex enzyme hydrolysis. Afterward, a 3 kDa molecular weight cut-off (MWCO) was used for centrifugation, and then fractionation was carried out using Strong Cation Exchange (SCX) Chromatography. The quantification of antioxidant activity was performed using the DPPH test. The results showed that the thermolysin hydrolysate (IC50 1.48 ± 0.92 mg/mL) and the combination of trypsin-thermolysin hydrolysates (IC50 1.37 ± 0.84 mg/mL) had the highest antioxidant activity. The fractionation procedure with strong cation exchange (SCX) yielded fraction C from thermolysin hydrolysates, which exhibited the most significant antioxidant activity with an IC50 value of 0.47 ± 0.02 mg/mL. The fraction 4 derived from trypsin-thermolysin hydrolysates exhibited the most potent antioxidant activity (IC50: 0.34 ± 0.002 mg/mL). No previous reports have been made about the antioxidant properties of the hydrolysate fractions derived from C. formosanum proteins. Consequently, these findings could serve as a foundation for exploring peptides that have potential therapeutic sources for reducing oxidative stress.

Diagonal Chromatography for the Isolation of O-Linked β-N-acetyl-D-Glucosaminylated (O-GlcNAcylated) Peptides in HeLa Cell Extracts

Protein O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a dynamic post-translational modification process that plays an essential role in biological activities. Growing evidence indicates that aberration of O-GlcNAcylation is associated with various diseases, e.g. diabetes, neurological diseases, and cancers. However, the mechanistic studies of O-GlcNAcylation are lagging behind other post-translational modifications due to its extremely low abundance, limited analytical tools, and specificity. Herein, diagonal strong cation exchange chromatography was applied to enrich the O-GlcNAc glycosylated peptides prior to mass spectrometric analysis by liquid chromatography/ion trap tandem mass spectrometry (LC–MS/MS). In this strategy, the O-GlcNAcylated peptides were first enzymatically labeled with an azide-modified galactosamine (GalNAz) and fractionated by strong cation exchange (SCX) chromatography. Tris(carboxyethyl)phosphine (TCEP) reduces the azido group in GalNAz-modified peptides to a primary amine group. TCEP-induced reduction of GalNAz-modified peptides was separated from unmodified peptides by diagonal SCX. By reversed-phase LC–MS/MS analysis of secondary SCX fractions, O-GlcNAcylated peptides were isolated and identified from the mixtures of O-GlcNAc-modified and unmodified peptides in HeLa cell extract. A total of 250 O-GlcNAcylation sites on 215 proteins were identified. Therefore, this novel method could be a potential tool for the isolation and site analysis of O-GlcNAc-modified peptides.

Correction of creatine-creatinine conversion during serum creatinine quantification by two-dimensional liquid chromatography and double-spike isotope dilution tandem mass spectrometry

Background and aimsDevelopment of a candidate reference method based on bidimensional liquid chromatography coupled to ESI-MS/MS and double spike isotope dilution for serum creatinine quantification capable of correcting for creatinine-creatine interconversion during sample pretreatment. Study of the impact of the creatine-creatinine interconversion during the analysis of human serum samples. Materials and methods13C1-creatinine and 13C2-creatine are added to the serum sample. Separation carried out by bidimensional liquid chromatography combining reversed phase and a strong cation exchange chromatography. The heart cut, containing creatine and creatinine, is automatically transferred to the second dimension. Quantification carried out by double spike isotope dilution tandem MS/MS. ResultsMinimization of spectral interferences and ion suppression due to matrix effects while increasing sample throughput compared to the direct coupling of cation exchange chromatography to the ESI source. Trueness of the method studied with the satisfactory analysis of two certified reference materials. Satisfactory intra- and inter-day precisions obtained analysing a serum pool and control sera. Analysis of 93 serum samples revealed negligible interconversions with no correlation with creatine levels. ConclusionsThe method provides adequate analytical figures of merit for serum creatinine determination according to CSLI guidelines. Negligible creatine-creatinine interconversion is promoted with the applied sample preparation procedure.

Reverse phase, ion exchange, HILIC and mix-mode chromatography for the determination of metformin and evogliptin in human plasma and pharmaceutical formulations

Separation and quantification of highly polar metformin (MTF) alone or in combination with dipeptidyl peptidase-4 inhibitors, such as evogliptin (EVG), sitagliptin, saxagliptin, vildagliptin, linagliptin and teneligliptin, specifically from human plasma and pharmaceutical products is still difficult in mostly preferred ODS based RP-HPLC techniques. Since, owing to weak retention of MTF in ODS, it elutes together with the biological fluid components and drug excipients. Therefore, in this study, alternative to ODS based RP-HPLC, comparatively less known analytical techniques like strong cation exchange chromatography (SCX-3), hydrophilic interaction liquid chromatography (HILIC) and mix-mode chromatography (MMC) were comprehensively evaluated for their potentials and limitations in simultaneous quantification of newly approved EVG and MTF combination (Valera-M). In results, prolonged application of SCX-3, exhibits irreproducible and irreversible retention of MTF and EVG. While in HILIC with Cyano column, excessive acetonitrile as eluent developed the precipitation of MTF and column back pressure. Comparatively, the Acclaimed® mix-mode HILIC-1, demonstrated much promising and reproducible results for MTF and EVG separation and importantly, it can be used either reverse phase or HILIC mode. Considering the overall benefits of Acclaimed® mix-mode HILIC-1, it was used in estimation of MTF and EVG from human plasma and pharmaceutical formulations.

Discovery and Characterization of a Dual-Function Peptide Derived from Bitter Gourd Seed Protein Using Two Orthogonal Bioassay-Guided Fractionations Coupled with In Silico Analysis.

The hydrolysate of bitter gourd seed protein, digested by the combined gastrointestinal proteases (BGSP-GPs), exhibited the most potent inhibition on angiotensin-I-converting enzyme (ACE) with an IC50 value of 48.1 ± 2.0 µg/mL. Using two independent bioassay-guided fractionations, fraction F5 from reversed-phase chromatography and fraction S1 from strong cation exchange chromatography exhibited the highest ACE inhibitory (ACEI) activity. Three identical peptides were simultaneously detected from both fractions and, based on the in silico appraisal, APLVSW (AW6) was predicted as a promising ACEI peptide. Their dipeptidyl peptidase-IV (DPP4) inhibitory (DPP4I) activity was also explored. The IC50 values of AW6 against ACE and DPP4 were calculated to be 9.6 ± 0.3 and 145.4 ± 4.4 µM, respectively. The inhibitory kinetics and intermolecular interaction studies suggested that AW6 is an ACE competitive inhibitor and a DPP4 non-competitive inhibitor. The quantities of AW6 in BGSP-GP hydrolysate, fractions F5 and S1, were also analyzed using liquid chromatography-tandem mass spectrometry. Notably, AW6 could resist hydrolysis in the human gastrointestinal tract according to the result of the simulated gastrointestinal digestion. To the best of our knowledge, this is the first discovery and characterization of a dual-function (ACEI and DPP4I activities) peptide derived from bitter gourd seed protein.

Highly efficient and chemoselective blocking of free amino group by ortho-phthalaldehyde (OPA) for comprehensive analysis of protein terminome

Herein, we introduced ortho-phthalaldehyde (OPA) for blocking free amino groups and established a simple and robust method for comprehensive profiling of protein terminome based on strong cation exchange chromatography (SCX) fractionation. With the highly efficient and chemoseletive amine-group blocking, we identified 2271 canonical human protein N-termini, 1650 canonical human protein C-termini, as well as 645 protein neo-N-termini from HeLa cells.

Optimization for Simultaneous Removal of Product/Process-Related Impurities of Peptide Fc-Fusion Protein Using Cation Exchange Chromatography

Fc fusion proteins are used as therapeutic agents with unique structures by combining the Fc domain of an antibody with other active proteins, cytokines, and enzymes. Peptide Fc-fusion proteins are complex fusion molecules that possess a structure different from that of monoclonal antibodies (mAbs) and are difficult to express, thereby affecting their quality. Many product/process-related impurities generated during the production of peptide Fc-fusion proteins pose a risk to the robustness of pre-existing three-column platforms for the purification of mAbs. Thus, we first evaluated the effect of pH, conductivity, and dynamic binding capacity (DBC; g of product per liter of resin) on the separation of host cell protein (HCP) and high molecular weight (HMW) and low molecular weight (LMW) proteins in strong cation exchange chromatography and then established an operating range using the design of experiments (DoE). Based on our studies, the optimal removal rates of HCP and HMW were achieved under the following conditions: 8 CV of wash buffer, 20–23 g/L of resin DBC, and an elution buffer conductivity of 63–66 mS/cm. The conductivity of the wash buffer used to remove the LMW was 50 mS/cm. In addition, reproducibility was confirmed by scaling up two batches using the Fractogel® EMD SO3− (M) resin. As a result of confirming with a validated test method in all batches, >55% yield, >98.2% purity, and >27% HCP reduction rate were satisfied. The cation exchanger exhibited an acceptable step yield and effectively reduced product/process-related impurities within the established range.

First Report of Screening of Novel Angiotensin-I Converting Enzyme Inhibitory Peptides Derived from the Red Alga Acrochaetium sp.

ACE inhibitors generated from food proteins have recently become the most well-known subclass of bioactive peptides, and their bio-functionality can be a potential alternative to natural bioactive food components and synthetic drugs. The bioactivities of Acrochaetium sp., the red alga used in this investigation, have never been reported before. Screening of bioactive peptides from Acrochaetium sp. as ACE inhibitors were hydrolyzed with various proteolytic enzymes. Protein hydrolysates were fractionated separately using reversed phased (RP) and strong cation exchange (SCX) chromatography and identified as VGGSDLQAL (VL-9) using α-chymotrypsin. It comes from Phycoerythrin (PE), an abundant protein in a primarily red alga. The peptide VL-9 shows the ACE inhibitory activity with IC50 value 433.1 ± 1.08µM. The inhibition pattern showed VL-9 as a non-competitive inhibitor. Molecular docking simulation proved that VL-9 was non-competitive inhibition due to the interaction peptide and ACE was not in the catalytic site. Moreover, VL-9 derived from Acrochaetium sp. is a natural bioactive peptide that is safer and available for food protein; also, the ACE inhibitory peptide derived from Acrochaetium sp. could be the one alternative resource to develop functional food for combating hypertension.

Correlative N-glycan and charge variant analysis of cetuximab expressed in murine, chinese hamster and human expression systems

A well-defined and controlled glycosylation pattern is important to maintain quality and safety of therapeutic proteins. Glycosylation is strongly dependent on the host cell line used for recombinant protein expression. Cetuximab, which is produced in mouse myeloma cells has been shown to harbour Fab glycans, which contain non-human like features and hence, can potentially cause an immunogenic response in patients. In light of the advent of biosimilar and biobetter development, we produced cetuximab variants in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. A combination of orthogonal chromatographic modes such as hydrophilic interaction, size exclusion and strong cation exchange chromatography with various detection strategies was employed to characterise the three different cetuximab variants and to compare the in-house produced HEK and CHO variants with the reference drug product. While Fc galactosylation and sialic acid content of the drug product and the HEK variant were highly similar, the CHO product showed lower galactosylation on Fc glycans and a comparatively low sialic acid content in the Fab region. The elevated high-mannose content of CHO cetuximab also suggests potential rapid clearence from circulation. The combination of multiple chromatographic separation modes has proven powerful for the characterisation of expression system dependent protein quality attributes such as N-glycosylation.

A Strong Cation Exchange Chromatography Protocol for Examining N-Terminal Proteoforms.

Especially in eukaryotes, the N-terminal acetylation status of a protein reveals translation initiation sites and substrate specificities and activities of N-terminal acetyltransferases (NATs). Here, we discuss a bottom-up proteomics protocol for the enrichment of N-terminal peptides via strong cation exchange chromatography. This protocol is based on depleting internal tryptic peptides from proteome digests through their retention on strong cation exchangers, leaving N-terminally acetylated/blocked peptides enriched among the nonretained peptides. As such, one can identify novel N-terminal proteoforms and quantify the degree of N-terminal protein acetylation.

Creating optimized peptide libraries for AMP discovery via PepSAVI-MS.

Antimicrobial peptides (AMPs) are host defense peptides with a range of functions/activities/modes of action that are ubiquitously expressed across all forms of life. Continued discovery of novel AMPs presents exciting opportunities to address evolving resistance to existing treatments in multiple fields, including agricultural pathogens/pests as well as antimicrobial and chemotherapeutics for human health. However, typical discovery methods including bioassay-guided fractionation and genome mining generally lack the capacity for robust AMP discovery in non-model/unsequenced organisms. PepSAVI-MS (Statistically guided bioactive peptides prioritized via mass spectrometry) was developed as an AMP discovery approach that addresses some of the limitations associated with these standard methods. PepSAVI-MS is a multi-pronged pipeline that includes peptide library creation, bioactivity screening, LC-MS analysis, and statistical modeling for putative AMP identification. The original implementation of PepSAVI-MS outlined strategies for the fractionation of plant extracts with strong cation exchange chromatography (SCX). Herein, we elaborate on recent improvements to peptide library creation through the use of orthogonal fractionation methods, specifically crude SCX chromatography and reversed-phase liquid chromatography (RPLC). This optimization of the "peptide library creation" step has demonstrated improvements for processing and AMP identifications via PepSAVI-MS.

Integrated Strategy for Unbiased Profiling of the Histidine Phosphoproteome.

Histidine phosphorylation (pHis), which plays a key role in signal transduction in bacteria and lower eukaryotes, has been shown to be involved in tumorigenesis. Due to its chemical instability, substoichiometric properties, and lack of specific enrichment reagents, there is a lack of approaches for specific and unbiased enrichment of pHis-proteins/peptides. In this study, an integrated strategy was established and evaluated as an unbiased tool for exploring the histidine phosphoproteome. First, taking advantage of the lower charge states of pHis-peptides versus the non-modified naked peptides at weak acid solution (∼pH 2.7), strong cation exchange (SCX) chromatography was used to differentiate modified and non-modified naked peptides. Furthermore, selective enrichment of the pHis-peptide was performed by applying Cu-IDA beads enrichment. Finally, stable isotope dimethyl labeling was introduced to guarantee high-confidence assignment of pHis-peptides. Using this integrated strategy, 563 different pHis-peptides (H = 1) in 385 proteins were identified from HeLa lysates. Motif analysis revealed that pHis prefers hydrophobic amino acids and has the consensus motif-HxxK, which covered the reports from different approaches. Thus, our method may provide an unbiased and effective tool to reveal histidine phosphoproteome and to study the biological process and function of histidine phosphorylation.

Antioxidant Activity from the Enzymatic Hydrolysates of Chlorella sorokiniana and Its Potential Peptides Identification in Combination with Molecular Docking Analysis

Chlorella sorokiniana is an edible microalga known for its high protein content with a balanced amino acid composition, nutritional value, beneficial health effect, and natural antioxidant. The enzymatic assays was used to extract the peptides of C. sorokiniana which was an uncommon method to test the antioxidant activity. In this research, protein of C. sorokiniana was extracted, purified, and hydrolyzed in several enzymes and kept at 37°C for 16h. Hence, enzymatic hydrolysate <3kDA was fractionated into 11 portions (C0%, C10%, to C100%) by using offline Strong Cation Exchange Chromatography (SCX) and their antioxidant activity was tested using DPPH (2.2-diphenyl-1-picrylhydrazyl) radical scavenging assay. The results indicated that C80%, contributed to the highest free DPPH scavenging on C. sorokiniana hydrolysate with the inhibition of 22.04%. Furthermore, to find the candidate peptides, this fraction was injected into LC-MS/MS for characterization of it's DPPH inhibitation. LSSATSAPS (m/z 1638,78) and AGLYGHPQTQEE (m/z 1328.59) are peptides that were identified and confirmed by LC-MS/MS. The molecular docking study was conducted to provide the binding simulation between these peptides and the ROS1 as the receptor. In conclusion, our results suggested that the aforementioned peptides were attached to ROS1 binding site and contributed to its potential antioxidant activity.

Strong cation exchange chromatography: a new option for concurrent quantification of saxagliptin and metformin

Strong cation exchange chromatography: a new option for concurrent quantification of saxagliptin and metformin

Purification, characterization, and mode of action of Paracin 54, a novel bacteriocin against Staphylococci.

Staphylococci belong to conditionally pathogenic bacteria, and the pathogenicity of Staphylococcus aureus is the strongest among them. Enterotoxin produced by it can contaminate food and cause food poisoning. Bacteriocin is a kind of polypeptide with antibacterial activity synthesized by some bacteria during metabolism. In this study, we report on purification, characterization, and mode of action of the bacteriocin named Paracin 54, produced by Lactobacillus paracasei ZFM54. Paracin 54 was purified by precipitation with 80% ammonium sulfate, strong cation-exchange chromatography, G-25 gel column, and reversed-phase high-performance liquid chromatography (HPLC). The molecular weight of Paracin 54 (5718.1843Da) was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Paracin 54 showed broad-spectrum inhibitory activity. It had a strong inhibitory effect on Staphylococci with minimum inhibitory concentration values of 3.00-4.50μg/mL. Paracin 54 was heat-stable and active only in acidic pH range (2-6). After treatment with proteases, the activity was lost. The results of mode of action showed Paracin 54 damaged the cell membrane and cell wall of Staphylococcus aureus, and then the cytoplasm leaked out, leading to death of the bacteria. These properties make Paracin 54 a promising candidate to prevent the growth of spoilage bacteria and control food poisoning caused by Staphylococci. KEY POINTS: • Paracin 54 was purified from Lactobacillus paracasei ZFM54 with good biochemical characteristics. • Paracin 54 had a strong effect against Staphylococci, making it a promising preservative to prevent the growth of Staphylococci in food. • The mode of action of Paracin 54 on Staphylococcus aureus was revealed.

Simultaneous Monitoring of Monoclonal Antibody Variants by Strong Cation-Exchange Chromatography Hyphenated to Mass Spectrometry to Assess Quality Attributes of Rituximab-Based Biotherapeutics.

Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of these proteoforms, novel approaches employing strong cation-exchange (SCX) high-performance liquid chromatography (HPLC) hyphenated to mass spectrometry (MS) using a pH gradient of volatile salts have been developed in recent years. Here, we apply an established SCX-HPLC-MS method to characterize and compare two rituximab-based biotherapeutics, the originator MabThera® and its Indian copy product Reditux™. The study assessed molecular differences between the two drug products in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. Overall, MabThera® and Reditux™ displayed differences at the molecular level. MabThera® showed a higher degree of galactosylated and sialylated glycoforms, while Reditux™ showed increased levels of oligomannose and afucosylated glycoforms. Moreover, the two drug products showed differences in terms of basic variants such as C-terminal lysine and N-terminal truncation, present in Reditux™ but not in MabThera®. This study demonstrates the capability of this fast SCX-HPLC-MS approach to compare different drug products and simultaneously assess some of their quality attributes.