ABSTRACT
In multiple myeloma (MM) disease, malignant plasma cells produce excessive quantities of a monoclonal immunoglobulin (Ig), known as M-protein. M-protein levels are measured in the serum of patients with MM using electrophoresis techniques to determine the response to treatment. However, therapeutic monoclonal antibodies, such as isatuximab, may confound signals using electrophoresis assays. We developed a robust assay based on immunocapture and liquid chromatography coupled to high-resolution mass spectrometry (IC-HPLC-HRMS) in order to eliminate this interference. Following immunocapture of Ig and free light chains (LC) in serum, heavy chains (HC) and LC were dissociated using dithiothreitol, sorted by liquid chromatography and analyzed using HRMS (Q-Orbitrap). This method allowed the M-proteins to be characterized and the signals from isatuximab and M-proteins to be discriminated. As M-protein is specific to each patient, no standards were available for absolute quantification. We therefore used alemtuzumab (an IgG kappa mAb) as a surrogate analyte for the semiquantification of M-protein in serum. This assay was successfully validated in terms of selectivity/specificity, accuracy/precision, robustness, dilution linearity, and matrix variability from 10.0 to 200 µg/mL in human serum. This method was used for clinical assessment of samples and eliminated potential interference due to isatuximab when monitoring patients with MM.
Subject(s)
Antibodies, Monoclonal , Immunoglobulins/blood , Multiple Myeloma/diagnosis , Antibodies, Monoclonal, Humanized , Chromatography, High Pressure Liquid , Chromatography, Liquid , Humans , Mass SpectrometryABSTRACT
Mechanisms of unassisted delivery of RNA therapeutics, including inhibitors of microRNAs, remain poorly understood. We observed that the hepatocellular carcinoma cell line SKHEP1 retains productive free uptake of a miR-21 inhibitor (anti-miR-21). Uptake of anti-miR-21, but not a mismatch (MM) control, induces expression of known miR-21 targets (DDAH1, ANKRD46) and leads to dose-dependent inhibition of cell growth. To elucidate mechanisms of SKHEP1 sensitivity to anti-miR-21, we conducted an unbiased shRNA screen that revealed tumor susceptibility gene 101 (TSG101), a component of the endosomal sorting complex required for transport (ESCRT-I), as an important determinant of anti-proliferative effects of anti-miR-21. RNA interference-mediated knockdown of TSG101 and another ESCRT-I protein, VPS28, improved uptake of anti-miR-21 in parental SKHEP1 cells and restored productive uptake to SKHEP1 clones with acquired resistance to anti-miR-21. Depletion of ESCRT-I in several additional cancer cell lines with inherently poor uptake resulted in improved activity of anti-miR-21. Finally, knockdown of TSG101 increased uptake of anti-miR-21 by cancer cells in vivo following systemic delivery. Collectively, these data support an important role for the ESCRT-I complex in the regulation of productive free uptake of anti-miRs and reveal potential avenues for improving oligonucleotide free uptake by cancer cells.
Subject(s)
Endosomal Sorting Complexes Required for Transport/metabolism , MicroRNAs/antagonists & inhibitors , Neoplasms/metabolism , Oligonucleotides/metabolism , Animals , Biological Transport , Cell Line, Tumor , DNA-Binding Proteins/physiology , Endosomal Sorting Complexes Required for Transport/antagonists & inhibitors , Endosomal Sorting Complexes Required for Transport/physiology , Female , Humans , Mice, SCID , MicroRNAs/metabolism , Neoplasms/genetics , Transcription Factors/physiologyABSTRACT
Regulation of the extent of immune responses is a requirement to maintain self-tolerance and limit inflammatory processes. CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells play a role in regulation. The Foxp3 transcription factor is considered a dominant regulator for Treg cell development and function. Foxp3 function itself is directly regulated by multiple posttranslational modifications that occur in response to various external stimuli. The Foxp3 protein is a component of several dynamic macromolecular regulatory complexes. The complexes change constituents over time and through different signals to regulate the development and function of regulatory T cells. Here we identified a mechanism regulating Foxp3 level and activity that operates through discrete phosphorylation. The Pim-2 kinase can phosphorylate Foxp3, leading to decreased suppressive functions of Treg cells. The amino-terminal domain of Foxp3 is modified at several sites by Pim-2 kinase. This modification leads to altered expression of proteins related to Treg cell functions and increased Treg cell lineage stability. Treg cell suppressive function can be up-regulated by either pharmacologically inhibiting Pim-2 kinase activity or by genetically knocking out Pim-2 in rodent Treg cells. Deficiency of Pim-2 activity increases murine host resistance to dextran sodium sulfate-induced colitis in vivo, and a Pim-2 small molecule kinase inhibitor also modified Treg cell functions. Our studies define a pathway for limiting the regulation of Foxp3 function because the Pim-2 kinase represents a potential therapeutic target for modulating the Treg cell suppressive activities in controlling immune responses.
Subject(s)
Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , T-Lymphocytes, Regulatory/immunology , Amino Acid Sequence , Animals , Mice , Mice, Knockout , Molecular Sequence Data , Phosphorylation , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/geneticsABSTRACT
Elevated blood concentrations of homocysteine have been well established as a risk factor for cardiovascular diseases and neuropsychiatric diseases, yet the etiologic relationship of homocysteine to these disorders remains poorly understood. Protein N-homocysteinylation has been hypothesized as a contributing factor; however, it has not been examined globally owing to the lack of suitable detection methods. We recently developed a selective chemical method to label N-homocysteinylated proteins with a biotin-aldehyde tag followed by Western blotting analysis, which was further optimized in this study. We then investigated the variation of protein N-homocysteinylation in plasma from rats on a vitamin B12 deficient diet. Elevated "total homocysteine" concentrations were determined in rats with a vitamin B12 deficient diet. Correspondingly, overall levels of plasma protein N-homocysteinylation displayed an increased trend, and furthermore, more pronounced and statistically significant changes (e.g., 1.8-fold, p-value: 0.03) were observed for some individual protein bands. Our results suggest that, as expected, a general metabolic correlation exists between "total homocysteine" and N-homocysteinylation, although other factors are involved in homocysteine/homocysteine thiolactone metabolism, such as the transsulfuration of homocysteine by cystathionine ß-synthase or the hydrolysis of homocysteine thiolactone by paraoxonase 1 (PON1), may play more significant or direct roles in determining the level of N-homocysteinylation.
Subject(s)
Blood Proteins/metabolism , Homocysteine/blood , Hyperhomocysteinemia/blood , Plasma/metabolism , Protein Processing, Post-Translational , Vitamin B 12 Deficiency/blood , Animals , RatsABSTRACT
Intensive microbial growth typically observed in laboratory rarely occurs in nature. Because of severe nutrient deficiency, natural populations exhibit near-zero growth (NZG). There is a long-standing controversy about sustained NZG, specifically whether there is a minimum growth rate below which cells die or whether cells enter a non-growing maintenance state. Using chemostat with cell retention (CCR) of Pseudomonas putida, we resolve this controversy and show that under NZG conditions, bacteria differentiate into growing and VBNC (viable but not non-culturable) forms, the latter preserving measurable catabolic activity. The proliferating cells attained a steady state, their slow growth balanced by VBNC production. Proteomic analysis revealed upregulated (transporters, stress response, self-degrading enzymes and extracellular polymers) and downregulated (ribosomal, chemotactic and primary biosynthetic enzymes) proteins in the CCR versus batch culture. Based on these profiles, we identified intracellular processes associated with NZG and generated a mathematical model that simulated the observations. We conclude that NZG requires controlled partial self-digestion and deep reconfiguration of the metabolic machinery that results in the biosynthesis of new products and development of broad stress resistance. CCR allows efficient on-line control of NZG including VBNC production. A well-nuanced understanding of NZG is important to understand microbial processes in situ and for optimal design of environmental technologies.
Subject(s)
Pseudomonas putida/growth & development , Bacterial Proteins/metabolism , Kinetics , Microbial Viability , Mutation , Proteomics , Pseudomonas putida/cytology , Pseudomonas putida/genetics , Pseudomonas putida/metabolismABSTRACT
The formation of isoaspartyl residues (isoAsp or isoD) via either aspartyl isomerization or asparaginyl deamidation alters protein structure and potentially biological function. This is a spontaneous and nonenzymatic process, ubiquitous both in vivo and in nonbiological systems, such as in protein pharmaceuticals. In almost all organisms, protein L-isoaspartate O-methyltransferase (PIMT, EC2.1.1.77) recognizes and initiates the conversion of isoAsp back to aspartic acid. Additionally, alternative proteolytic and excretion pathways to metabolize isoaspartyl-containing proteins have been proposed but not fully explored, largely due to the analytical challenges for detecting isoAsp. We report here the relative quantitation and site profiling of isoAsp in urinary proteins from wild type and PIMT-deficient mice, representing products from excretion pathways. First, using a biochemical approach, we found that the total isoaspartyl level of proteins in urine of PIMT-deficient male mice was elevated. Subsequently, the major isoaspartyl protein species in urine from these mice were identified as major urinary proteins (MUPs) by shotgun proteomics. To enhance the sensitivity of isoAsp detection, a targeted proteomic approach using electron transfer dissociation-selected reaction monitoring (ETD-SRM) was developed to investigate isoAsp sites in MUPs. A total of 38 putative isoAsp modification sites in MUPs were investigated, with five derived from the deamidation of asparagine that were confirmed to contribute to the elevated isoAsp levels. Our findings lend experimental evidence for the hypothesized excretion pathway for isoAsp proteins. Additionally, the developed method opens up the possibility to explore processing mechanisms of isoaspartyl proteins at the molecular level, such as the fate of protein pharmaceuticals in circulation.
Subject(s)
Isoaspartic Acid/metabolism , Protein D-Aspartate-L-Isoaspartate Methyltransferase/metabolism , Proteins/analysis , Proteomics , Tandem Mass Spectrometry , Animals , Chromatography, High Pressure Liquid , Deamination , Mice , Mice, Knockout , Peptides/analysis , Protein D-Aspartate-L-Isoaspartate Methyltransferase/deficiency , Protein D-Aspartate-L-Isoaspartate Methyltransferase/genetics , Proteins/metabolismABSTRACT
Protein biomarkers are critical for diagnosis, prognosis, and treatment of disease. The transition from protein biomarker discovery to verification can be a rate limiting step in clinical development of new diagnostics. Liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS) is becoming an important tool for biomarker verification studies in highly complex biological samples. Analyte enrichment or sample fractionation is often necessary to reduce sample complexity and improve sensitivity of SRM for quantitation of clinically relevant biomarker candidates present at the low ng/mL range in blood. In this paper, we describe an alternative method for sample preparation for LC-SRM MS, which does not rely on availability of antibodies. This new platform is based on selective enrichment of proteotypic peptides from complex biological peptide mixtures via isoelectric focusing (IEF) on a digital ProteomeChip (dPC) for SRM quantitation using a triple quadrupole (QQQ) instrument with an LC-Chip (Chip/Chip/SRM). To demonstrate the value of this approach, the optimization of the Chip/Chip/SRM platform was performed using prostate specific antigen (PSA) added to female plasma as a model system. The combination of immunodepletion of albumin and IgG with peptide fractionation on the dPC, followed by SRM analysis, resulted in a limit of quantitation of PSA added to female plasma at the level of â¼1-2.5 ng/mL with a CV of â¼13%. The optimized platform was applied to measure levels of PSA in plasma of a small cohort of male patients with prostate cancer (PCa) and healthy matched controls with concentrations ranging from 1.5 to 25 ng/mL. A good correlation (r(2) = 0.9459) was observed between standard clinical ELISA tests and the SRM-based assay. Our data demonstrate that the combination of IEF on the dPC and SRM (Chip/Chip/SRM) can be successfully applied for verification of low abundance protein biomarkers in complex samples.
Subject(s)
Blood Proteins/analysis , Chromatography, Liquid/instrumentation , Isoelectric Focusing/instrumentation , Mass Spectrometry/instrumentation , Protein Array Analysis/instrumentation , Amino Acid Sequence , Animals , Biomarkers/blood , Blood Proteins/isolation & purification , Cattle , Chromatography, Liquid/methods , Female , Humans , Immunosorbent Techniques , Isoelectric Focusing/methods , Limit of Detection , Linear Models , Male , Mass Spectrometry/methods , Molecular Sequence Data , Peptide Fragments/analysis , Prostate-Specific Antigen/analysis , Prostate-Specific Antigen/isolation & purification , Proteomics/instrumentation , Proteomics/methods , Reproducibility of Results , Trypsin/chemistryABSTRACT
Seasonal influenza vaccines confer protection against specific viral strains but have restricted breadth that limits their protective efficacy. The H1 and H3 subtypes of influenza A virus cause most of the seasonal epidemics observed in humans and are the major drivers of influenza A virus-associated mortality. The consequences of pandemic spread of COVID-19 underscore the public health importance of prospective vaccine development. Here, we show that headless hemagglutinin (HA) stabilized-stem immunogens presented on ferritin nanoparticles elicit broadly neutralizing antibody (bnAb) responses to diverse H1 and H3 viruses in nonhuman primates (NHPs) when delivered with a squalene-based oil-in-water emulsion adjuvant, AF03. The neutralization potency and breadth of antibodies isolated from NHPs were comparable to human bnAbs and extended to mismatched heterosubtypic influenza viruses. Although NHPs lack the immunoglobulin germline VH1-69 residues associated with the most prevalent human stem-directed bnAbs, other gene families compensated to generate bnAbs. Isolation and structural analyses of vaccine-induced bnAbs revealed extensive interaction with the fusion peptide on the HA stem, which is essential for viral entry. Antibodies elicited by these headless HA stabilized-stem vaccines neutralized diverse H1 and H3 influenza viruses and shared a mode of recognition analogous to human bnAbs, suggesting that these vaccines have the potential to confer broadly protective immunity against diverse viruses responsible for seasonal and pandemic influenza infections in humans.
Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza Vaccines/immunology , Primates/immunology , Animals , Antibodies, Viral/biosynthesis , Antibodies, Viral/chemistry , Antigen-Antibody Complex/chemistry , Broadly Neutralizing Antibodies/biosynthesis , Broadly Neutralizing Antibodies/chemistry , COVID-19 , Ferritins/chemistry , Ferritins/immunology , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Humans , Influenza Vaccines/administration & dosage , Influenza Vaccines/chemistry , Influenza, Human/immunology , Influenza, Human/virology , Macaca fascicularis , Models, Molecular , Nanoparticles/chemistry , Pandemics , Primates/virology , Protein Structure, Quaternary , SARS-CoV-2 , Translational Research, BiomedicalABSTRACT
A ubiquitous yet underappreciated protein post-translational modification, isoaspartic acid (isoAsp, isoD, or beta-Asp), generated via the deamidation of asparagine or isomerization of aspartic acid in proteins, plays a diverse and crucial role in aging, as well as autoimmune, cancer, neurodegeneration, and other diseases. In addition, formation of isoAsp is a major concern in protein pharmaceuticals, as it may lead to aggregation or activity loss. The scope and significance of isoAsp have, up to now, not been fully explored, as an unbiased screening of isoAsp at low abundance remains challenging. This difficulty is due to the subtle difference in the physicochemical properties between isoAsp and Asp, e.g., identical mass. In contrast, endoprotease Asp-N (EC 3.4.24.33) selectively cleaves aspartyl peptides but not the isoaspartyl counterparts. As a consequence, isoaspartyl peptides can be differentiated from those containing Asp and also enriched by Asp-N digestion. Subsequently, the existence and site of isoaspartate can be confirmed by electron transfer dissociation (ETD) mass spectrometry. As little as 0.5% of isoAsp was detected in synthetic beta-amyloid and cytochrome c peptides, even though both were initially assumed to be free of isoAsp. Taken together, our approach should expedite the unbiased discovery of isoAsp.
Subject(s)
Chromatography, High Pressure Liquid/methods , Isoaspartic Acid/analysis , Mass Spectrometry/methods , Metalloendopeptidases/metabolism , Amino Acid Sequence , Amyloid/chemistry , Cytochromes c/chemistry , Electron Transport , Molecular Sequence DataABSTRACT
In the biotechnology industry, the generation of incorrectly folded recombinant proteins, either from an E.coli expression system or from an overexpressed CHO cell line (disulfide scrambling), is often a great concern as such incorrectly folded forms may not be completely removed in the final product. Thus, significant efforts have been devoted to map disulfide bonds to ensure drug quality. Similar to ECD, disulfide bond cleavages are preferred over peptide backbone fragmentation in ETD. Thus, an online LC-MS strategy combining collision-induced dissociation (CID-MS(2)), electron-transfer dissociation (ETD-MS(2)), and CID of an isolated product ion derived from ETD (MS(3)) has been used to characterize disulfide-linked peptides. Disulfide-linked peptide ions were identified by CID and ETD fragmentation, and the disulfide-dissociated (or partially dissociated) peptide ions were characterized in the subsequent MS(3) step. The online LC-MS approach is successfully demonstrated in the characterization of disulfide linkages of recombinant human growth hormone (Nutropin), a therapeutic monoclonal antibody, and tissue plasminogen activator (Activase). The characterization of disulfide-dissociated or partially dissociated peptide ions in the MS(3) step is important to assign the disulfide linkages, particularly, for intertwined disulfide bridges and the unexpected disulfide scrambling of tissue plasminogen activator. The disulfide-dissociated peptide ions are shown to be obtained either directly from the ETD fragmentation of the precursors (disulfide-linked peptide ions) or indirectly from the charge-reduced species in the ETD fragmentation of the precursors. The simultaneous observation of disulfide-linked and disulfide-dissociated peptide ions with high abundance not only provided facile interpretation with high confidence but also simplified the conventional approach for determination of disulfide linkages, which often requires two separate experiments (with and without chemical reduction). The online LC-MS with ETD methodology represents a powerful approach to aid in the characterization of the correct folding of therapeutic proteins.
Subject(s)
Antibodies, Monoclonal/analysis , Disulfides/analysis , Human Growth Hormone/analysis , Recombinant Proteins/analysis , Tissue Plasminogen Activator/analysis , Amino Acid Sequence , Antibodies, Monoclonal/chemistry , Chromatography, Liquid/methods , Cysteine/analysis , Cysteine/chemistry , Disulfides/chemistry , Human Growth Hormone/chemistry , Humans , Molecular Sequence Data , Recombinant Proteins/chemistry , Tandem Mass Spectrometry/methods , Tissue Plasminogen Activator/chemistryABSTRACT
Elevated blood levels of homocysteine (Hcy), hyperhomocysteinemia or homocystinuria, have been associated with various diseases and conditions. Homocysteine thiolactone (Hcy TL) is a metabolite of Hcy and reacts with amine groups in proteins to form stable amides, homocystamides, or N-homocysteinylated proteins. It has been proposed that protein N-homocysteinylation contributes to the cytotoxicity of elevated Hcy. Due to its heterogeneity and relatively low abundance, detection of this posttranslational modification remains challenging. On the other hand, the gamma-aminothiol group in homocystamides imparts different chemical reactivities than the native proteins. Under mildly acidic conditions, gamma-aminothiols irreversibly and stoichiometrically react with aldehydes to form stable 1,3-thiazines, whereas the reversible Schiff base formation between aldehydes and amino groups in native proteins is markedly disfavored due to protonation of amines. As such, we have developed highly selective chemical methods to derivatize N-homocysteinylated proteins with various aldehyde tags, thereby facilitating the subsequent analyses. For instance, fluorescent or biotin tagging coupled with gel electrophoresis permits quantification and global profiling of complex biological samples, such as hemoglobin and plasma from rat, mouse and human; affinity enrichment with aldehyde resins drastically reduces sample complexity. In addition, different reactivities of lysine residues in hemoglobin toward Hcy TL were observed.
Subject(s)
Aldehydes/chemistry , Blotting, Western/methods , Hemoglobins/chemistry , Homocysteine/analysis , Luminescent Measurements/methods , Amino Acid Sequence , Animals , Fluorescent Dyes/chemistry , Homocysteine/blood , Homocysteine/chemistry , Humans , Mass Spectrometry , Mice , Rats , Rhodamines/chemistryABSTRACT
Modern proteomic research frequently relies upon separation of proteins in a polyacrylamide gel matrix followed by in-gel enzymatic digestion and extraction of peptides for subsequent analysis by MS. In this work, we propose a novel semi-automated method of mechanical processing of gel bands by passing these bands through a specially designed centrifugal device termed a Gel Shredder prior to digestion and extraction of peptides. Such a device allows integrated washing, destaining and shredding of gel bands into uniform blocks of controlled size, approximately 150-300 microm, prior to the enzymatic digestion and extraction of peptides. Shredding into uniform blocks increases the surface area of the gel pieces and promotes improved gel rehydration, allowing improved diffusion of the proteolytic enzymes and solvent into the gel lattice. We demonstrate that the new method substantially reduces the time spent on tedious manual handling of gel bands, while minimizing the risk of sample contamination. The performance of the Gel Shredder has been compared with a conventional in-gel digestion protocol using several standard proteins and a complex proteomic sample in terms of relative quantitation by either MALDI-TOF/TOF or nanoLC-ESI IT-Fourier transformation ion cyclotron resonance MS. It is shown that significant time savings and improved peptide recovery can be obtained for many proteins using the Gel Shredder compared with the traditional in-gel digestion protocol.
Subject(s)
Centrifugation , Electrophoresis, Polyacrylamide Gel , Mass Spectrometry , Peptide Fragments , Peptide Mapping , Adipose Tissue/chemistry , Animals , Chromatography, Liquid , Databases, Protein , Equipment Design , Mice , Peptide Fragments/analysis , Peptide Fragments/metabolism , Peptide Mapping/instrumentation , Peptide Mapping/methods , Proteins/analysis , Proteins/metabolism , Proteomics/instrumentation , Proteomics/methods , Trypsin/metabolismABSTRACT
Halogenated furanones, a group of natural products initially isolated from marine red algae, are known to inhibit bacterial biofilm formation, swarming, and quorum sensing. However, their molecular targets and the precise mode of action remain elusive. Herein, we show that a naturally occurring brominated furanone covalently modifies and inactivates LuxS (S-ribosylhomocysteine lyase, EC 4.4.1.21), the enzyme which produces autoinducer-2 (AI-2).
Subject(s)
Anti-Bacterial Agents/chemistry , Bacterial Proteins/metabolism , Bromine/chemistry , Carbon-Sulfur Lyases/metabolism , Enzyme Inhibitors/chemistry , Furans/chemistry , Rhodophyta/chemistry , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/chemistry , Carbon-Sulfur Lyases/chemistry , Enzyme Inhibitors/pharmacology , Furans/pharmacologyABSTRACT
Seasonal influenza vaccines represent a positive intervention to limit the spread of the virus and protect public health. Yet continual influenza evolution and its ability to evade immunity pose a constant threat. For these reasons, vaccines with improved potency and breadth of protection remain an important need. We previously developed a next-generation influenza vaccine that displays the trimeric influenza hemagglutinin (HA) on a ferritin nanoparticle (NP) to optimize its presentation. Similar to other vaccines, HA-nanoparticle vaccine efficacy is increased by the inclusion of adjuvants during immunization. To identify the optimal adjuvants to enhance influenza immunity, we systematically analyzed TLR agonists for their ability to elicit immune responses. HA-NPs were compatible with nearly all adjuvants tested, including TLR2, TLR4, TLR7/8, and TLR9 agonists, squalene oil-in-water mixtures, and STING agonists. In addition, we chemically conjugated TLR7/8 and TLR9 ligands directly to the HA-ferritin nanoparticle. These TLR agonist-conjugated nanoparticles induced stronger antibody responses than nanoparticles alone, which allowed the use of a 5000-fold-lower dose of adjuvant than traditional admixtures. One candidate, the oil-in-water adjuvant AF03, was also tested in non-human primates and showed strong induction of neutralizing responses against both matched and heterologous H1N1 viruses. These data suggest that AF03, along with certain TLR agonists, enhance strong neutralizing antibody responses following influenza vaccination and may improve the breadth, potency, and ultimately vaccine protection in humans.
Subject(s)
Adjuvants, Immunologic/pharmacology , Antibodies, Neutralizing/immunology , Influenza Vaccines/immunology , Adjuvants, Immunologic/chemistry , Animals , Female , HEK293 Cells , Hemagglutination Inhibition Tests , Hemagglutinins , Humans , Macaca mulatta , Mice, Inbred BALB C , Nanoparticles , Toll-Like Receptors/agonistsABSTRACT
Isoaspartate formation is a ubiquitous post-translation modification arising from spontaneous asparagine deamidation or aspartate isomerization. The formation of isoaspartate inserts a methylene group into the protein backbone, generating a "kink", and may drastically alter protein structure and function, thereby playing critical roles in a myriad of biological processes, human diseases, and protein pharmaceutical development. Herein, we report a chemo-enzymatic detection method for the isoaspartate protein, which in particular allows the affinity enrichment of isoaspartate-containing proteins. In the initial step, protein isoaspartate methyltransferase selectively converts isoaspartates into the corresponding methyl esters. Subsequently, the labile methyl ester is trapped by strong nucleophiles in aqueous solutions, such as hydrazines to form hydrazides. The stable hydrazide products can be analyzed by standard proteomic techniques, such as matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry. Furthermore, the chemical trapping step allows us to introduce several tagging strategies for product identification and quantification, such as UV-vis and fluorescence detection through a dansyl derivative. Most significantly, the hydrazide product can be enriched by affinity chromatography using aldehyde resins, thus drastically reducing sample complexity. Our method hence represents the first technique for the affinity enrichment of isoaspartyl proteins and should be amendable to the systematic and comprehensive characterization of isoaspartate, particularly in complex systems.
Subject(s)
Hydrazines/chemistry , Isoaspartic Acid/analysis , Protein D-Aspartate-L-Isoaspartate Methyltransferase/chemistry , Proteins/chemistry , Chromatography, Affinity , Spectrometry, Fluorescence , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform InfraredABSTRACT
A highly precise, automatic and rapid method for quantification of puerarin in canine and human plasma using an on-line solid-phase extraction (SPE) column switching procedure combined with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS) was developed. The eluent of SPE column consisted of acetonitrile/methanol/0.1% formic acid (25/25/50) at a flow rate of 0.2mLmin(-1). Puerarin was analyzed by a linear ion trap mass spectrometer, LTQ-MS, operating in the negative ion and selective reaction monitoring (SRM) acquisition mode. Method validation results demonstrated that the linear calibration curve covered a wide range of 0.39-400.00ngmL(-1), the correlation coefficients (r(2)) were above 0.999. The lower limit of detection (LLOD) with the signal-to-noise (S/N) ratio higher than 12 was 0.39ngmL(-1). The intra- and inter-batch precisions were less than 7.61% and 6.42%, respectively. The accuracy was well within the accept limit. The on-line SPE column switching HPLC-MS system was applied to pharmacokinetic (PK) study of puerarin after a single orally dose in beagles. And the optimum conditions were successfully utilized to quantify puerarin in human plasma, which indicated the feasibility and the reliability of this method for application in preclinical and clinical PK studies of isoflavone drugs.
Subject(s)
Isoflavones/blood , Isoflavones/pharmacokinetics , Vasodilator Agents/blood , Vasodilator Agents/pharmacokinetics , Animals , Calibration , Chromatography, High Pressure Liquid , Dogs , Humans , Indicators and Reagents , Male , Quality Control , Reference Standards , Reproducibility of Results , Solvents , Tandem Mass SpectrometryABSTRACT
Large-scale bioprocessing is key to the successful manufacturing of a biopharmaceutical. However, cell viability and productivity are often lower in the scale-up from laboratory to production. In this study, we analyzed CHO cells, which showed lower percent viabilities and productivity in a 5-KL production scale bioreactor compared to a 20-L bench-top scale under seemingly identical process parameters. An increase in copper concentration in the media from 0.02 µM to 0.4 µM led to a doubling of percent viability in the production scale albeit still at a lower level than the bench-top scale. Combined metabolomics and proteomics revealed the increased copper reduced the presence of reactive oxygen species (ROS) in the 5-KL scale process. The reduction in oxidative stress was supported by the increased level of glutathione peroxidase in the lower copper level condition. The excess ROS was shown to be due to hypoxia (intermittent), as evidenced by the reduction in fibronectin with increased copper. The 20-L scale showed much less hypoxia and thus less excess ROS generation, resulting in little to no impact to productivity with the increased copper in the media. The study illustrates the power of 'Omics in aiding in the understanding of biological processes in biopharmaceutical production.
Subject(s)
Batch Cell Culture Techniques/methods , Fibronectins/metabolism , Metabolomics/methods , Proteomics/methods , Reactive Oxygen Species/metabolism , Animals , Bioreactors , CHO Cells , Cell Hypoxia , Cell Proliferation , Cell Survival , Copper , Cricetulus , HumansABSTRACT
A goal in recombinant protein production using Chinese hamster ovary (CHO) cells is to achieve both high specific productivity and high cell density. Addition of glucose to the culture media is necessary to maintain both cell growth and viability. We varied the glucose concentration in the media from 5 to 16 g/L and found that although specific productivity of CHO-DG44 cells increased with the glucose level, the integrated viable cell density decreased. To examine the biological basis of these results, we conducted a discovery proteomic study of CHO-DG44 cells grown under batch conditions in normal (5 g/L) or high (15 g/L) glucose over 3, 6, and 9 days. Approximately 5,000 proteins were confidently identified against an mRNA-based CHO-DG44 specific proteome database, with 2,800 proteins quantified with at least two peptides. A self-organizing map algorithm was used to deconvolute temporal expression profiles of quantitated proteins. Functional analysis of altered proteins suggested that differences in growth between the two glucose levels resulted from changes in crosstalk between glucose metabolism, recombinant protein expression, and cell death, providing an overall picture of the responses to high glucose environment. The high glucose environment may enhance recombinant dihydrofolate reductase in CHO cells by up-regulating NCK1 and down-regulating PRKRA, and may lower integrated viable cell density by activating mitochondrial- and endoplasmic reticulum-mediated cell death pathways by up-regulating HtrA2 and calpains. These proteins are suggested as potential targets for bioengineering to enhance recombinant protein production.
Subject(s)
Culture Media/pharmacology , Glucose/metabolism , Glucose/pharmacology , Proteome/analysis , Proteome/drug effects , Animals , Bioreactors , CHO Cells , Cricetinae , Cricetulus , Culture Media/chemistry , Culture Media/metabolism , Glucose/chemistry , Proteome/metabolism , ProteomicsABSTRACT
OBJECTIVE: To study the compatibility of composite herbal medicines of the Ling Gui Zhu Gan Decoction. METHOD: Ethanol extract test solutions of the different combinations were prepared according to the orthogonal layout L16(4(5)). Pharmacologic experiments, such as the time of surviving of mice in shortage of oxygen in regular air pressure, the antagonizing effect on arrhythmia induced by chloroform and diuresis were carried out with the solutions. Variance analysis, canonical correlation and stepwise regression analysis were applied to interrelate the amount of each drug and the pharmacologic data. RESULT: The results confirmed that Fuling and Guizhi are the basis, while Baizhuand Gancao are the adjuvans, which is conformed to the theory of TCM. CONCLUSION: This study provides a significant try for studying the compatibility of composite herbal medicines.
Subject(s)
Drugs, Chinese Herbal , Plant Extracts , Plants, Medicinal , Animals , Anti-Arrhythmia Agents/therapeutic use , Diuretics/pharmacology , Drug Combinations , Drugs, Chinese Herbal/isolation & purification , Drugs, Chinese Herbal/therapeutic use , Female , Hypoxia/drug therapy , Male , Medicine, Chinese Traditional , Mice , Phytotherapy , Plant Extracts/isolation & purification , Plant Extracts/therapeutic use , Plants, Medicinal/chemistry , Ventricular Fibrillation/drug therapyABSTRACT
Pseudomonas species are capable to proliferate under diverse environmental conditions and thus have a significant bioremediation potential. To enhance our understanding of their metabolic versatility, this study explores the changes in the proteome and physiology of Pseudomonas putida F1 resulting from its growth on benzoate, a moderate toxic compound that can be catabolized, and citrate, a carbon source that is assimilated through central metabolic pathways. A series of repetitive batch cultivations were performed to ensure a complete adaptation of the bacteria to each of these contrasting carbon sources. After several growth cycles, cell growth stabilized at the maximum level and exhibited a reproducible growth profile. The specific growth rates measured for benzoate (1.01 ± 0.11 h-1) and citrate (1.11 ± 0.12 h-1) were similar, while a higher yield was observed for benzoate (0.6 and 0.3 g cell mass per g of benzoate and citrate, respectively), reflecting the different degrees of carbon reduction in the two substrates. Comparative proteomic analysis revealed an enrichment of several oxygenases/dehydrogenases in benzoate-grown cells, indicative of the higher carbon reduction of benzoate. Moreover, the upregulation of all 14 proteins implicated in benzoate degradation via the catechol ortho-cleavage pathway was observed, while several stress-response proteins were increased to aid cells to cope with benzoate toxicity. Unexpectedly, citrate posed more challenges than benzoate in the maintenance of pH homeostasis, as indicated by the enhancement of the Na+/H+ antiporter and carbonic anhydrase. The study provides important mechanistic insights into Pseudomonas adaptation to varying carbon sources that are of great relevance to bioremediation efforts.