Weak Cation Exchange Chromatography Research Articles

In-line buffer exchange in the coupling of Protein A chromatography with weak cation exchange chromatography for the determination of charge variants of immunoglobulin G derived from chinese hamster ovary cell cultures

Immunoglobulin G (IgG) is the most common monoclonal antibody (mAb) grown for therapeutic applications. While IgG is often selectively isolated from cell lines using protein A (ProA) chromatography, this is only a stepping stone for complete characterization. Further classification can be obtained from weak cation exchange chromatography (WCX) to determine IgG charge variant distributions. The charge variants of monoclonal antibodies can influence the stability and efficacy in vivo, and deviations in charge heterogeneity are often cell-specific and sensitive to upstream process variability. Current methods to characterize IgG charge variants are often performed off-line, meaning that the IgG eluate from the ProA separation is collected, diluted to adjust the pH, and then transferred to the WCX separation, adding time, complexity, and potential contamination to the sample analysis process. More recently, reports have appeared to streamline this separation using in-line two-dimensional liquid chromatography (2D-LC). Presented here is a novel, 2D-LC coupling of ProA in the first dimension (1D) and WCX in the second dimension (2D) chromatography. As anticipated, the initial direct column coupling proved to be challenging due to the pH incompatibility between the mobile phases for the two stages. To solve the solvent compatibility issue, a size exclusion column was placed in the switching valve loop of the 2D-LC instrument to act as a means for the on-line solvent exchange. The efficacy of the methodology presented was confirmed through a charge variant determination using the NIST monoclonal antibody standard (NIST mAb), yielding correct acidic, main, and basic variant compositions. The methodology was employed to determine the charge variant profile of IgG from an in-house cultured Chinese hamster ovary (CHO) cell supernatant. It is believed that this methodology can be easily implemented to provide higher-throughput assessment of IgG charge variants for process monitoring and cell line development.

Multiattribute Monitoring of Aggregates and Charge Variants of Monoclonal Antibody through Native 2D-SEC-MS-WCX-MS.

Monitoring of critical quality attributes such as size and charge-related heterogeneities is essential for biopharmaceutical manufacturers. Size-exclusion chromatography (SEC) is the preferred analytical technique for the quantification of aggregates and fragments in the product, whereas weak-cation exchange chromatography (WCX) is widely used for the characterization of charge variants of biotherapeutic products, in particular monoclonal antibodies (mAbs). Multiattribute monitoring offers the ability to monitor these attributes in a single run flow using two-dimensional liquid chromatography (2D-LC). Typically, in this approach, only the second-dimension samples are directly analyzed through mass spectrometry, as the first dimension has limitations concerning direct coupling with mass spectrometry. In the present study, a novel 2D-SEC-MS/WCX-MS workflow has been proposed, in which chromatography of both dimensions (D1 and D2) was directly coupled with mass spectrometry, through which size-related and charge-related variants of monoclonal antibody mAb A were analyzed simultaneously in their native form. In comparison to stand-alone SEC and WCX methods, this method enables simultaneous analysis of size and charge variants in a single workflow without manual intervention, allowing analysis of low abundant variants. Further, this method has 75% less sample requirement and a shorter analysis time (25 min vs 90 min) when size and charge variants were analyzed individually. The proposed native 2D-LC-MS workflow was used to analyze a stressed sample of mAb A, in which D1 analysis revealed the presence of aggregates (8-20%), which were primarily dimers, whereas D2 analysis showed an increment in acidic variants (9-21%).

Multiattribute Monitoring of Charge-Based Heterogeneity of Recombinant Monoclonal Antibodies Using 2D HIC-WCX-MS.

Charged heterogeneity of monoclonal antibody (mAb) products is regarded as a critical quality attribute (CQA) depending on its impact on the safety and efficacy profile of the product. Hence, manufacturers are expected to perform a comprehensive characterization of the charge heterogeneity to ensure that the manufactured product meets its specifications. Further, monitoring is also expected during the product lifecycle to demonstrate consistency in product quality. However, conventional analytical methods for characterization of hydrophobic and charge variants are nonvolatile salt-based and require manual fraction collection and desalting steps before analysis through mass spectrometry can be performed. In the present study, a workflow of a two-dimensional liquid chromatography method using mass spectrometry (MS)-compatible buffers coupled with native mass spectrometry was performed to characterize hydrophobic variants in the first dimension and charge variants in the second dimension without any need for manual fractionation. This novel two-dimensional (2D) hydrophobic interaction chromatography (HIC)-weak cation-exchange chromatography (WCX)-MS workflow identified 10 variants in mAb A, out of which 2 variants are exclusive to the 2D orthogonal method. Similarly, for mAb B, a total of 11 variants are identified, including 5 variants exclusive to the 2D orthogonal workflow. When compared to stand-alone, HIC resolved only 4 variants for both mAbs and WCX resolved 7 variants for mAb A and 6 variants for mAb B. In addition, the proposed method allows direct characterization of hydrophobic/charge variant peaks through native mass spectrometry in a single-run workflow.

In situphotoinitiated fabrication of phosphorylcholine-functionalized polyhedral oligomeric silsesquioxane hybrid monolithic column for mixed-mode capillary electrochromatography

A phosphorylcholine-functionalized POSS hybrid monolithic column was synthesizedviaUV curing. It exhibits hydrophilic interaction and weak cation exchange chromatography retention mechanism for the separation of typical polar and charged compounds.

Sample displacement chromatography of monoclonal antibody charge variants and aggregates.

The rise of biosimilar monoclonal antibodies has renewed the interest in monoclonal antibody (mAb) charge variants composition and separation. The sample displacement chromatography (SDC) has the potential to overcome the low separation efficiency and productivity associated with bind-elute separation of mAb charge variants. SDC in combination with weak cation exchanging macroporous monolithic chromatographic column was successfully implemented for a separation of charge variants and aggregates of monoclonal IgG under overloading conditions. The charge variants composition was at-line monitored by a newly developed, simple and fast analytical method, based on weak cation exchange chromatography. It was proven that basic charge variants acted as displacers of IgG molecules with lower pI, when the loading was performed 1 to 1.5pH unit below the pI of acidic charge variants. The efficiency of the SDC process is flow rate independent due to a convection-based mass transfer on the macroporous monolith. The productivity of the process at optimal conditions is 35mg of purified IgG fraction per milliliters of monolithic support with 75-80% recovery. As such, an SDC approach surpasses the standard bind-elute separation in the productivity for a factor of 3, when performed on the same column. The applicability of the SDC approach was confirmed for porous particle-based column as well, but with 1.5 lower productivity compared to the monoliths.

Capture and purification of an untagged nanobody by mixed weak cation chromatography and cation exchange chromatography

Nanobodies (Nbs) are single-domain antibodies, which have potential application value in tumor-targeted therapy, immunotherapy, diagnostic probe, and molecular imaging. Typically, Nbs are captured by affinity chromatography via the addition of specific fusion tags at their N or C terminus. Nerve growth factor (NGF), which regulates the growth and development of peripheral and central neurons, maintains neuronal survival and plays a key role in both arthritis and acute and chronic pain. In this study, a method for capture and purification of an untagged Nb (anti-NGF Nb) by mixed weak cation chromatography and cation exchange chromatography was established. First, pH 4.0–5.0 was demonstrated to be the optimal loading condition for Capto MMC to capture anti-NGF by the design of experiments (DOE). Furthermore, high purity and yield products can be obtained at laboratory scale and commercial production scale by adjusting the protein pH. Additionally, direct capture of anti-NGF Nb using Capto MMC without adjusting anti-NGF Nb harvest pH was investigated. The anti-NGF Nb captured by Capto MMC was 67.2% yield, 94.5% monomer purity, and host cell protein (HCP) was reduced from 74,931 ppm to 484 ppm. The anti-NGF Nb that were further purified using Capto S ImpAct achieved 84.5% yield and 99.2% purity and 77 ppm of HCP. The scaling-up process was consistent with the results of the optimized process, further demonstrating the feasibility of this method. This outcome provides a highly promising and competitive alternative to affinity chromatography-based processing strategies for Nbs.

P1615IDENTIFICATION OF URINARY PROTEOMIC PROFILE OF PATIENTS WITH CHRONIC ALLOGRAFT NEPHROPATHY

Abstract Background and Aims Chronic allograft nephropathy (CAN) is a major complication that occurs post-transplantation. At present, the diagnosis of chronic allograft nephropathy (CAN) is based on renal biopsy. Therefore, there is an ultimate need to identify more specific and sensitive noninvasive methods for the early diagnosis of CAN. Recently, proteomic-based modalities have been developed to discover biomarkers of CAN. Method Urine samples from 75 participants were collected. Participants were divided into 3 groups: Group I: 25 patients with chronic allograft nephropathy, group II: 25transplanted patients with stable renal functions, and group III: 25 healthy control subjects matched for age and sex. Each group was divided into training set and test set. Specimens were purified with magnetic beads-based weak cation exchange chromatography and analyzed in a MALDI-TOF MS. Results A Genetic Algorithm (GA) was used to set up the classification models. Five peaks represented the proteomic profile that differentiates between the CAN patients and the control group with sensitivity of 100%, specificity of 100%, recognition capability of 100 % and cross-validation91.7 % and five peaks differentiate between the transplant patients with normal renal functions and the control groups with sensitivity of96.8 %, specificity of 95.5 %, recognition capability of 98 % and cross-validation of 100 %. Conclusion We identified a pattern for CAN and transplant patients with normal renal functions by proteomic profiling using MALDI-TOF-MS and magnetic beads.

Identification of urinary proteomic profile of patients with chronic allograft nephropathy

ABSTRACT Background and objectives Chronic allograft nephropathy (CAN) is a major complication that occurs post-transplantation. At present, the diagnosis of CAN is based on renal biopsy. Therefore, there is an ultimate need to identify more specific and sensitive noninvasive methods for the early diagnosis of CAN. Recently, proteomic-based modalities have been developed to discover biomarkers of CAN. Methods Urine samples from 75 participants were collected. Participants were divided into three groups: Group I: 25 patients with CAN, Group II: 25 transplanted patients with stable renal functions, and Group III: 25 healthy control subjects matched for age and sex. Each group was divided into training set and test set. Specimens were purified with magnetic beads-based weak cation exchange chromatography and analyzed using MALDI-TOF MS. Results A Genetic Algorithm (GA) was used to set up the classification models. Five peaks represented the proteomic profile that differentiates between the CAN patients and the control group with sensitivity of 100%, specificity of 100%, recognition capability of 100%, and cross-validation of 91.7% and five peaks differentiate between the transplant patients with normal renal functions and the control groups with sensitivity of 96.8%, specificity of 95.5%, recognition capability of 98%, and cross-validation of 100%. Interpretation and conclusions We identified a pattern for CAN and transplant patients with normal renal functions by proteomic profiling using MALDI-TOF-MS and magnetic beads.

Peptidome profiles in melamine diet-induced bladder stones in C57BL/6 mice

The aim of this research was to detect potential serum biomarkers of melamine diet-induced bladder stones in C57BL/6 mice. Magnetic bead-based weak cationexchange chromatography (MB-WCX) and matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) were employed to detect serum biomarkers in 10 mice fed a melamine diet and 10 control mice. Seventeen peaks (fold change>1.5) with a mass to charge (m/z) value of 1000–10,000 Da were detected in the two groups. Among the significant peaks, five were upregulated and the other 12 were downregulated in the model group. Among the upregulated peaks, 2954.49 and 1710.49 were found to correspond to the peptide regions of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8(Ndufα8) and basigin, respectively, by liquid chromatography with electrospray ionization and tandem triple quadrupole mass spectrometry(LC-ESI-MS/MS). Western blot analysis was used to detect the expression of Ndufα8 and basigin in another 10 model mice and 10 control mice. The western blot results confirmed the LC-ESI-MS/MS data. The expression of serum basigin and Ndufα8 was partly dependent the concentration of melamine, but no time dependence. In conclusion, Ndufα8 and basigin may be potential serum biomarkers for the detection of melamine diet-induced bladder stones in C57BL/6 mice

Characterization of bispecific and mispaired IgGs by native charge-variant mass spectrometry

Bispecific antibodies (BsAbs) combine the specificities of two antibodies to target different antigens. While production and in vivo assembly of BsAb using a single-cell host is less resource intensive compared to two-cell production, in particular mispaired light chain antibody species are a unique and undesired side product. Different pairing strategies may be employed to increase correct BsAb content, however, the potential presence of mispaired species must be both identified and quantified. Quantitative analysis by mass spectrometry is limited to chromatographic separation of the antibody assemblies due to their similar biophysical properties. This is particularly challenging for distinguishing correctly paired and light chain-scrambled mispaired antibodies which have the same molecular weight. Traditionally, imaged capillary isoelectric focusing (iCIEF) has been employed for analyzing charge-based heterogeneities, but this technique lacks resolution. Here we describe a powerful analytical platform using native weak cation exchange chromatography coupled to a high-resolution mass spectrometer, charge variant mass spectrometry (CV-MS), to characterize bispecific and mispaired antibody species. We investigate elution order through analytical methods and molecular modeling in an effort to understand the intrinsic charge, size and shape differences of these molecules. Although isoelectric points of the variants are similar, localized charge patches are hypothesized to offer sufficient micro-variations to be able to resolve and then identify isobars using CV-MS.

Non-affinity purification of a nanobody by void-exclusion anion exchange chromatography and multimodal weak cation exchange chromatography

Multimodal chromatography resins have been reported to be excellent alternatives to classical absorbents in the purification of antibodies. In this study, void-exclusion anion exchange chromatography (VEAX) and electropositive multimodal chromatography (Capto MMC) were integrated for tag-free nanobody (2D5) purification. VEAX was systematically characterized for its potential use in the initial 2D5 separation. VEAX achieved a 89.9% reduction in host cell proteins (HCPs), a 99.6% reduction in DNA, a 99.9% reduction in endotoxins, and a 100% reduction in aggregates from periplasmic proteins, with a 2D5 recovery of near 100% and a purity enhancement from 3.5% to 25.0%. Subsequently, Capto MMC chromatography was seamlessly incorporated with direct protein loading for further purification. The recovery of this step was 80.5%. The entire two-step purification platform yielded 2D5 with 16 ppm of HCPs, 127 ppb of DNA, 35 EU/mL of endotoxins, with a total protein recovery of 80.5%. The purity of 2D5 was 98.5%. Moreover, this method had no effect on the affinity of 2D5. Meanwhile, the established method was also used to purify a nanobody against PD-L1 (KPU) with 95.8% purity, >99% recovery, 55 ppm of HCPs, 289 ppb of DNA and 128 EU/mL of endotoxins. For the first time, this study reports a simplified, seamless and non-affinity purification platform for the purification of a tag-free nanobody, which provides a competitive alternative for the conventional affinity chromatography antibody purification strategy.

Characterization of recombinant monoclonal antibody charge variants using WCX chromatography, icIEF and LC-MS/MS

Charge heterogeneity is an important aspect of research into the development of monoclonal antibody drugs. In the present study, charge variants were separated into four fractions using weak cation exchange chromatography and were thoroughly analyzed using liquid chromatography–mass spectrometry at multiple levels. Molecular weight analysis of intact antibody and subunits confirmed the presence of heavy-chain leader sequences, light-chain leader sequences, dehydration, and cysteinylation. Peptide mapping of the fractions using different enzymes further localized the modified sites. Modified proportions identified at peptide level were compared with the purity detected by imaged capillary isoelectric focusing, the results showed that basic variant 1 consisted of cysteinylation and dehydration of asparagine, and basic variant 2 fully accounted for the N-terminal leader sequence of the heavy chain. About 14.8% of the acidic variant can be explained by N-terminal leader sequences in the light chain, and 18% of the acidic variant was demonstrated to be deamidation of asparagine in the heavy chain. There was approximately 54.2% of the acidic variant still cannot be explained. It was hypothesized that those acidic variants that have not yet been identified are an ensemble of molecules with slight molecular weight differences or the same molecular weight but different structures.

Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS

Top-down proteomics is an emerging analytical strategy to characterize combinatorial protein post-translational modifications (PTMs). However, sample complexity and small mass differences between chemically closely related proteoforms often limit the resolution attainable by separations employing a single liquid chromatographic (LC) principle. In particular, for ultramodified proteins like histones, extensive and time-consuming fractionation is needed to achieve deep proteoform coverage. Herein, we present the first online nanoflow comprehensive two-dimensional liquid chromatography (nLC×LC) platform top-down mass spectrometry analysis of histone proteoforms. The described two-dimensional LC system combines weak cation exchange chromatography under hydrophilic interaction LC conditions (i.e., charge- and hydrophilicity-based separation) with reversed phase liquid chromatography (i.e., hydrophobicity-based separation). The two independent chemical selectivities were run at nanoflows (300 nL/min) and coupled online with high-resolution mass spectrometry employing ultraviolet photodissociation (UVPD-HRMS). The nLC×LC workflow increased the number of intact protein masses observable relative to one-dimensional approaches and allowed characterization of hundreds of proteoforms starting from limited sample quantities (∼1.5 μg).

Online Removal of Sodium Dodecyl Sulfate via Weak Cation Exchange in Liquid Chromatography-Mass Spectrometry Based Proteomics.

Biological research often requires the use of sodium dodecyl sulfate (SDS) to solubilize protein samples; however, this detergent is not compatible with direct mass spectrometry (MS) analysis. Here, we report an online high-throughput proteomics method that permits standard in-solution digestion of SDS-containing samples followed by direct liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis using weak cation-exchange chromatography (WCX). This approach, called the online removal of sodium dodecyl sulfate (Online reSDS), exploits the properties of WCX in a highly organic and mildly acidic medium to retain positively charged peptides by both hydrophilic interaction and electrostatic attraction while simultaneously repelling negative SDS molecules. This method was optimized to successfully analyze complex samples that contain up to 1% of SDS. Furthermore, online reSDS improves the identification of peptides with post-translational modifications (PTMs), such as deamidation and phosphorylation, without preliminary enrichment. In conclusion, we show that reSDS can facilitate research in proteomics by allowing the use of SDS in a wide range of LC-MS/MS applications with simplified sample-processing procedures.

Preparation and evaluation of a hydrophilic interaction and cation-exchange chromatography stationary phase modified with 2-methacryloyloxyethyl phosphorylcholine

In this work, 2-methacryloyloxyethyl phosphorylcholine (MPC) was used as a ligand to prepare a novel mixed-mode chromatography (MMC) stationary phase by the thiol-ene click reaction onto silica (MPC-silica). It was found that this MPC-silica showed the retention characteristics of hydrophilic interaction chromatography (HILIC) and weak cation exchange chromatography (WCX) under suitable mobile phase conditions. In detail, acidic and basic hydrophilic compounds and puerarin from pueraria were separated quickly with HILIC mode. Meanwhile, six standard proteins were allowed to reach baseline separation in WCX mode, and protein separation from egg white was also achieved with this mode. In addition, reduced/denatured lysozyme could be refolded with the MPC-silica column. In the meantime, the MPC-silica has been applied for refolding with simultaneous purification of recombinant human Delta-like1-RGD (rhDll1-RGD) expressed in Escherichia coli. The results show that the mass recovery and purity of rhDll1-RGD could reach 63.4% and 97% by one step, respectively. Furthermore, the reporter assay results demonstrated that refolded with simultaneously purified rhDll1-RGD could efficiently activate the signalling pathway in a dose-dependent manner. In general, this MPC-silica has good resolution and selectivity in the separation of polar compounds and protein samples in different high-performance liquid chromatography (HPLC) modes, and it successfully achieved refolding with simultaneous purification of denatured protein.

Insights into the generation of monoclonal antibody acidic charge variants during Chinese hamster ovary cell cultures.

Charge variation is one of the most important heterogeneities during monoclonal antibody (mAb) manufacturing and this study presents insights into the generation of acidic charge variants during cell culture processes. Since acidic variants generate both intracellularly and extracellularly, main charge fraction collected by weak cation exchange chromatography (WCX) was incubated in harvested cell supernatant (HCS) to simulate and investigate the extracellular process firstly. It is found that the main fraction was degraded rapidly into acidic variants rather than basic variants extracellularly, and the degradation sites were located in both Fab and Fc fragments indicated by papain digestion. Besides, certain process parameters were investigated as their potential roles in the extracellular process. As a result, media composition showed significant influence on degradation while culture time point did not, suggesting that the extracellular process was a spontaneous process without enzyme catalysis. Additionally, kinetics study reveals that the extracellular process was a pseudo first-order reaction. The E app value (21.59kcal/mol) estimated from the Arrhenius equation suggests that the extracellular degradation might be mainly attributed to asparagine deamidation. Furthermore, we established an acidic variants generation model, indicating that the extracellular process plays a dominant role in modulating the final acidic variant level. This study provides better understanding for controlling product heterogeneity in mAb manufacturing.

Identification of multiple sources of the acidic charge variants in an IgG1 monoclonal antibody.

Charge variants, especially acidic charge variants, of recombinant monoclonal antibodies are the major critical quality attributes in the biotechnology industry due to their potential influence on stability and biological activity. The chemical properties of the acidic charge variants have been challenging to fully characterize, and it is critical for process development and optimization. To completely understand the multiple sources of acidic charge variants, the major charge forms of an IgG1 monoclonal antibody were firstly isolated and then analyzed by a battery of characterization tools. It was found that various degrees of disulfidebond reduction, the deamination of HC-T8 Asn84 and HC-T35 Asn388 and aggregation account for the majority of acidic charge heterogeneity and the terminal galactosylation content was in relation to the acidic charge heterogeneity. The correlation between acidic charge heterogeneity and galactosylation content was further explored by weak cation exchange chromatography with the use of β1-4 galactosidase digestion. The results showed that galactosylation was not the source of the acidic charge variants per se. Meanwhile, to gain insights into the impact on binding affinity of monoclonal antibody to IgE and FcRn, charge variants were also analyzed by competitive ELISA and surface plasmon resonance, respectively. All isolated charge variants had similar affinity binding to IgE and FcRn binding relative to the starting material.

Application Value of Mass Spectrometry in the Differentiation of Benign and Malignant Liver Tumors.

BackgroundDifferentiation of malignant from benign liver tumors remains a challenging problem. In recent years, mass spectrometry (MS) technique has emerged as a promising strategy to diagnose a wide range of malignant tumors. The purpose of this study was to establish classification models to distinguish benign and malignant liver tumors and identify the liver cancer-specific peptides by mass spectrometry.Material/MethodsIn our study, serum samples from 43 patients with malignant liver tumors and 52 patients with benign liver tumors were treated with weak cation-exchange chromatography Magnetic Beads (MB-WCX) kits and analyzed by the Matrix-Assisted Laser Desorption Time of Flight Mass Spectrometry (MALDI-TOF-MS). Then we established genetic algorithm (GA), supervised neural networks (SNN), and quick classifier (QC) models to distinguish malignant from benign liver tumors. To confirm the clinical applicability of the established models, the blinded validation test was performed in 50 clinical serum samples. Discriminatory peaks associated with malignant liver tumors were subsequently identified by a qTOF Synapt G2-S system.ResultsA total of 27 discriminant peaks (p<0.05) in mass spectra of serum samples were found by ClinPro Tools software. Recognition capabilities of the established models were 100% (GA), 89.38% (SNN), and 80.84% (QC); cross-validation rates were 81.67% (GA), 81.11% (SNN), and 86.11% (QC). The accuracy rates of the blinded validation test were 78% (GA), 84% (SNN), and 84% (QC). From the 27 discriminatory peptide peaks analyzed, 3 peaks of m/z 2860.34, 2881.54, and 3155.67 were identified as a fragment of fibrinogen alpha chain, fibrinogen beta chain, and inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), respectively.ConclusionsOur results demonstrated that MS technique can be helpful in differentiation of benign and malignant liver tumors. Fibrinogen and ITIH4 might be used as biomarkers for the diagnosis of malignant liver tumors.

Proteins associated with EGFR-TKIs resistance in patients with non-small cell lung cancer revealed by mass spectrometry.

The present study aimed to identify potential serum biomarkers for predicting the clinical outcomes of patients with advanced non-small cell lung cancer (NSCLC) treated with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‑TKIs). A total of 61samples were collected and analyzed using the integrated approach of magnetic bead‑based weak cation exchange chromatography and matrix‑assisted laser desorption/ionization‑time of flight‑mass spectrometry. The Zhejiang University Protein Chip Data Analysis system was used to identify the protein spectra of patients that are resistant and sensitive to EGFR‑TKIs. Furthermore, a support vector machine was used to construct a predictive model with high accuracy. The model was trained using 46samples and tested with the remaining 15samples. In addition, the ExPASy Bioinformatics Resource Portal was used to search potential candidate proteins for peaks in the predictive model. Sevenmass/charge (m/z) peaks at3,264, 9,156, 9,172, 3,964, 9,451, 4,295 and 3,983Da, were identified as significantly different peaks between the EGFR‑TKIs sensitive and resistant groups. A predictive model was generated with three protein peaks at 3,264, 9,451 and 4,295Da(m/z). This three‑peak model was capable of distinguishing EGFR‑TKIs resistant patients from sensitive patients with a specificity of 80% and a sensitivity of 80.77%. Furthermore, in a blind test, this model exhibited a high specificity (80%) and a high sensitivity (90%). Apelin, TYRO protein tyrosine kinase‑binding protein and big endothelin‑1 may be potential candidates for the proteins identified with an m/z of3,264,9,451and 4,295Da, respectively. The predictive model used in the present study may provide an improved understanding of the pathogenesis of NSCLC, and may provide insights for the development of TKI treatment plans tailored to specific patients.

Retraction Note to: Effects of a Terrified-Sound Stress on Serum Proteomic Profiling in Mice.

The serum proteomic profiles of mice exposed to terrified-sound-induced stress and after stress release were investigated. Serum samples from 32 mice were divided into four groups (n = 8 each) and analyzed using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry techniques (MALDI-TOF MS) combined with magnetic bead-based weak cation-exchange chromatography. ClinProTools software identified several distinct markers that differed between the stressed and control groups and between the stress released and stressed released controls. Of 33 m/z peaks that differed among the four groups, 17 were significantly different (P < 0.05). Five peaks (m/z: 2793.37, 2924.86, 1979.90, 3492.49, 3880.24) showed significant differences in expression after exposure to terrified-sound stress and returned to control levels after stress release. These were sequence identified as peptide regions of dimethylaniline monooxygenase, myosin-9, uncharacterized protein in Rattus norvegicus, apolipoprotein C-I, and plasma serine protease inhibitor (Serpina 5). Our study provides the first evidence of significant changes in serum proteomic profiles in mice exposed to terrified-sound stress, which suggests that protein expression profiles are affected by the stress. Normal expression levels were restored after stress release, suggesting the activation of self-adjustment mechanisms for the recovery of protein expression levels altered by this stress.