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Monitoring of host cell proteins(HCPs)during the manufacturing of monoclonal antibodies(mAb)has become a critical requirement to provide effective and safe drug products.Enzyme-linked immunosor-bent assays are still the gold standard methods for the quantification of protein impurities.However,this technique has several limitations and does,among others,not enable the precise identification of pro-teins.In this context,mass spectrometry(MS)became an alternative and orthogonal method that de-livers qualitative and quantitative information on all identified HCPs.However,in order to be routinely implemented in biopharmaceutical companies,liquid chromatography-MS based methods still need to be standardized to provide highest sensitivity and robust and accurate quantification.Here,we present a promising MS-based analytical workflow coupling the use of an innovative quantification standard,the HCP Profiler solution,with a spectral library-based data-independent acquisition(DIA)method and strict data validation criteria.The performances of the HCP Profiler solution were compared to more con-ventional standard protein spikes and the DIA approach was benchmarked against a classical data-dependent acquisition on a series of samples produced at various stages of the manufacturing process.While we also explored spectral library-free DIA interpretation,the spectral library-based approach still showed highest accuracy and reproducibility(coefficients of variation＜10％)with a sensitivity down to the sub-ng/mg mAb level.Thus,this workflow is today mature to be used as a robust and straightforward method to support mAb manufacturing process developments and drug products quality control.
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Objective To identify the differentially expressed proteins in different liver tissues in the mouse model of alveolar echinococcosis using high-resolution mass spectrometry with data independent acquisition (DIA), and to identify the key proteins contributing to the pathogenesis of alveolar echinococcosis. Methods Protoscoleces were isolated from Microtus fuscus with alveolar echinococcosis and the experimental model of alveolar echinococcosis was established in female Kunming mice aged 6 to 8 weeks by infection with Echinococcus multilocularis protoscoleces. Mice were divided into the experimental and control groups, and animals in the experimental group was injected with approximately 3 000 protoscoleces, while mice in the control group were injected with the same volume of physiological saline. Mouse liver specimens were sampled from both groups one year post-infection and subjected to pathological examinations. In addition, the lesions (the lesion group) and peri-lesion specimens (the peri-lesion group) were sampled from the liver of mice in the experimental group and the normal liver specimens (the normal group) were sampled from mice in the control group for DIA proteomics analysis, and the differentially expressed proteins were subjected to bioinformatics analysis. Results A total of 1 020 differentially expressed proteins were identified between the lesion group and the normal group, including 671 up-regulated proteins and 349 down-regulated proteins, and 495 differentially expressed proteins were identified between the peri-lesion group and the normal group, including 327 up-regulated proteins and 168 down-regulated proteins. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that these differentially expressed proteins were involved in peroxisome, peroxisome proliferator-activated receptor (PPAR) and fatty acid degradation pathways, and the peroxisome and PPAR signaling pathways were found to correlate with liver injury. Several differentially expressed proteins that may contribute to the pathogenesis of alveolar echinococcosis were identified in these two pathways, including fatty acid binding protein 1 (Fabp1), Acyl-CoA synthetase long chain family member 1 (Acsl1), Acyl-CoA oxidase 1 (Acox1), Enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase (Ehhadh) and Acetyl-Coenzyme A acyltransferase 1B (Acaa1b), which were down-regulated in mice in the experimental group. Conclusion A large number of differentially expressed proteins are identified in the liver of the mouse model of alveolar echinococcosis, and Fabp1, Acsl1, Acox1, Ehhadh and Acaa1b may contribute to the pathogenesis of alveolar echinococcosis.
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Objective To identify the differentially expressed proteins in different liver tissues in the mouse model of cystic echinococcosis (CE), so as to provide insights into the research and development of therapeutic drugs targeting CE. Methods Female Kunming mice at ages of 6 to 8 weeks were randomly assigned into the CE group and the control group. Mice in the CE group were intraperitoneally infected with 2 000 Echinococcus multilocularis protoscoleces, while mice in the control group were injected with the same volume of physiological saline. All mice in both groups were sacrificed after breeding for 350 d, and the lesions (the lesion group) and peri-lesion specimens (the peri-lesion group) were sampled from the liver of mice in the CE group and the normal liver specimens (the normal group) were sampled from mice in the control group for data independent acquisition (DIA) proteomics analysis, and the differentially expressed proteins were subjected to Gene Ontology (GO) term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Results A total of 26 differentially expressed proteins were identified between the lesion group and the normal group and between the peri-lesion group and the normal group, including 8 up-regulated proteins and 18 down-regulated proteins. GO term enrichment analysis showed that these differentially expressed proteins were predominantly enriched in endoplasmic reticulum membrane (biological components), oxidoreductase activity (molecular function) and oxoacid metabolic process and monocarboxylic acid metabolic process (biological processes). KEGG pathway enrichment analysis revealed that the differentially expressed protein Acyl-CoA oxidase 1 (Acox1), which contributed to primary bile acid biosynthesis during the fatty acid oxidation, was involved in peroxisome signaling pathway, and the differentially expressed protein fatty acid binding protein 1 (Fabp1), which contributed to fatty acid transport, was involved in the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Conclusion Differentially expressed proteins are identified in the liver specimens between mouse models of CE and normal mice, and some differentially expressed proteins may serve as potential drug targets for CE.
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Lipidomics coverage improvement is essential for functional lipid and pathway construction.A powerful approach to discovering organism lipidome is to combine various data acquisitions,such as full scan mass spectrometry(full MS),data-dependent acquisition(DDA),and data-independent acquisition(DIA).Caenorhabditis elegans(C.elegans)is a useful model for discovering toxic-induced metabolism,high-throughput drug screening,and a variety of human disease pathways.To determine the lipidome of C.elegans and investigate lipid disruption from the molecular level to the system biology level,we used integrative data acquisition.The methyl-tert-butyl ether method was used to extract L4 stage C.elegans after exposure to triclosan(TCS),perfluorooctanoic acid,and nanopolystyrene(nPS).Full MS,DDA,and DIA integrations were performed to comprehensively profile the C.elegans lipidome by Q-Exactive Plus MS.All annotated lipids were then analyzed using lipid ontology and pathway analysis.We annotated up to 940 lipids from 20 lipid classes involved in various functions and pathways.The biological in-vestigations revealed that when C.elegans were exposed to nPS,lipid droplets were disrupted,whereas plasma membrane-functionalized lipids were likely to be changed in the TCS treatment group.The nPS treatment caused a significant disruption in lipid storage.Triacylglycerol,glycerophospholipid,and ether class lipids were those primarily hindered by toxicants.Finally,toxicant exposure frequently involved numerous lipid-related pathways,including the phosphoinositide 3-kinase/protein kinase B pathway.In conclusion,an integrative data acquisition strategy was used to characterize the C elegans lipidome,providing valuable biological insights into hypothesis generation and validation.
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Ensuring the removal of host cell proteins (HCPs) during downstream processing of recombinant pro-teins such as monoclonal antibodies (mAbs) remains a challenge.Since residual HCPs might affect product stability or safety,constant monitoring is required to demonstrate their removal to be below the regulatory accepted level of 100 ng/mg.The current standard analytical approach for this procedure is based on ELISA;however,this approach only measures the overall HCP content.Therefore,the use of orthogonal methods,such as liquid chromatography-mass spectrometry (LC-MS),has been established,as it facilitates the quantitation of total HCPs as well as the identification and quantitation of the indi-vidual HCPs present.In the present study,a workflow for HCP detection and quantitation using an automated magnetic bead-based sample preparation,in combination with a data-independent acquisi-tion (DIA) LC-MS analysis,was established.Employing the same instrumental setup commonly used for peptide mapping analysis of mAbs allows for its quick and easy implementation into pre-existing workflows,avoiding the need for dedicated instrumentation or personnel.Thereby,quantitation of HCPs over a broad dynamic range was enabled to allow monitoring of problematic HCPs or to track changes upon altered bioprocessing conditions.
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@#Most of the proteomics analysis methods based on tandem mass spectrometry rely on the matching scoring of actual spectrum and theoretical spectrum, the interference of a large number of co-eluting peptides could cause error in the identification and quantification of peptides and proteins. Peptide retention time prediction, as a auxiliary and verification index of the peptide, can transition the chromatographic behavior into stable independent time attributes, and improve the accuracy of the peptide identification. Prediction of peptide chromatographic retention in complex systems is also of great significance for optimizing proteomics determination conditions and improving the detection rate and repeatability of mass spectrometry data in data-independent acquisition. This review focused on the chromatographic retention prediction method of unmodified peptides and modified peptides, summarizes the content, characteristics and limitations of four types of peptide retention time prediction methods based on standardized indexes, peptide molecular models, amino acid residue parameters, and machine learning, as well as their applications in proteomics, with a prospect of their future.
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A new method of MS/MS~(ALL) was designed to sequentially record a MS~2 spectrum at each unit mass window through gas phase fractionation concept, so as to offer an opportunity for universal MS~2 spectral recording with direct infusion(DI). As a proof-of-concept, DI-MS/MS~(ALL) was applied for rapid chemome profiling of a famous herbal medicine named Lonicerae Japonicae Flos. After each MS~2 spectrum was correlated to its precursor ion, the structural annotation was conducted by applying well-defined mass cracking rules, matching the mass spectral data with literatures and referring to those accessible databases. As a result, a total of 54 components were identified from Lonicerae Japonicae Flos extract, including 21 phenolic acids, 13 flavonoids, 12 iridoids, 4 triterpenoids and 4 other compounds. Therefore, DI-MS/MS~(ALL) is a powerful tool for comprehensive, rapid qualitative analysis of chemical profiles of traditional Chinese medicine and other chemical components of complex systems.
Subject(s)
Chromatography, High Pressure Liquid , Drugs, Chinese Herbal , Lonicera , Plant Extracts , Tandem Mass SpectrometryABSTRACT
Objective To investigate the feasibility of differential expression proteins identification in peripheral blood mononuclear cells (PBMCs) of sepsis patients using Data Independent Acquisition liquid chromatography-mass spectrometry (DIA LC-MS). Methods Prospective studies were employed and targeted at 10 sepsis patients admitted to the Intensive Care Unit (ICU) of Shanghai Fifth People's Hospital Affiliated to Fudan University from April 2016 to July 2016. And 10 patients admitted to the ICU with similar age and sex that were not complicated with sepsis were served as the control group.The proteins from peripheral blood mononuclear cells in 10 sepsis patients and 10 control persons were analyzed using the latest DIA LC-MS technology and the skyline data extraction software; the model of data obtained from the above analysis was further discriminatorily analyzed with principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA); then the differential proteins of peripheral blood mononuclear cells were analyzed by Pathway analysis and GO analysis. The possible markers were identified by preliminary screening according to variable importance in the projection (VIP). The top 3 proteins (VIP > 1, P< 0.05) were verified by ELISA and their ROC curves were analyzed. Results Totally 1062 fragment ions were identified and 119 proteins were obtained. Among them, 31 proteins were up-regulated and 88 proteins down-regulated. Pathway analysis showed that carbon metabolism, platelet activation, bacterial invasion of epithelium, and complement coagulation cascade activation were participated in the development of sepsis. ELISA showed that significant difference of HMGB-1, MMP-8, and LCN2 in the expression of peripheral blood mononuclear cells in the sepsis and control people (P<0.01). The area under the ROC curve is greater than 0.85, which has good sensitivity and specificity. Conclusions DIA-MS is a compelling way for detecting differential expression proteins. PCA, PLSDA and OPLSDA are suitable for pattern recognition. The high expression of HMGB-1, NGAL and MMP-8 in immune cells may be the potential biomarker of the disease, which lays the foundation for research of early diagnosis and treatment of sepsis.
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Mass spectrometry is an important and powerful tool for protein quantification. With the in-depth development of quantitative proteomics, limitations of classic MS based quantification methods, such as complicated matrix interference and throughput/capacity limitation, start to appear. Recent progress of series novel MS based techniques provide effective solutions for the limitations of relative and absolute proteomic quantification, including synchronous precursor selection ( SPS ) , mass defect isobaric labeling, parallel reaction monitoring ( PRM) , multiplexing acquisition ( MSX) , and various novel data independent acquisition ( DIA) modes. Here we summarized the current limitations of quantitative proteomics, reviewed the latest MS based quantification approaches, and discussed the features and advantages of these novel techniques for quantitative proteomic application.
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Data independent acquisition ( DIA ) is a novel MS scan mode for quantitative proteomics, acquiring all precursors as well as fragments without any loss of low abundant ions, and breaks the throughput limitation of product ion quantification by high-resolution MS. Here we developed three DIA methods on quadrupole-linear ion trap-Orbitrap ( Q-qIT-OT ) Tribrid MS, classic DIA, as well as novel wide isolation window SIM scan ( WiSIM)-DIA and full scan-DIA ( Full MS-DIA) . Quantitative analysis of 10 low abundant peptides spiked in Hela cell digest was performed by the three methods for linearity, reproducibility and sensitivity evaluation. The results showed that the LOQs reached amol level ( 14-435 amol ) with good linearity and effective MS/MS confirmation. WiSIM-DIA utilizes ultra-high resolution SIM scan for quantification complementary with classic DIA. The isolation window of Full MS-DIA was down to 3 amu, and the data could be directly used for database searching, thus realizing the integration of data dependent acquisition ( DDA) and DIA, and avoiding the limitation of using spectra library.