ABSTRACT
The nucleolus is sensitive to stress and can orchestrate a chain of cellular events in response to stress signals. Despite being a growth factor, FGF2 has antiproliferative and tumor-suppressive functions in some cellular contexts. In this work, we investigated how the antiproliferative effect of FGF2 modulates chromatin-, nucleolus- and rDNA-associated proteins. The chromatin and nucleolar proteome indicated that FGF2 stimulation modulates proteins related to transcription, rRNA expression and chromatin-remodeling proteins. The global transcriptional rate and nucleolus area increased along with nucleolar disorganization upon 24â h of FGF2 stimulation. FGF2 stimulation induced immature rRNA accumulation by increasing rRNA transcription. The rDNA-associated protein analysis reinforced that FGF2 stimulus interferes with transcription and rRNA processing. RNA Pol I inhibition partially reversed the growth arrest induced by FGF2, indicating that changes in rRNA expression might be crucial for triggering the antiproliferative effect. Taken together, we demonstrate that the antiproliferative FGF2 stimulus triggers significant transcriptional changes and modulates the main cell transcription site, the nucleolus.
Subject(s)
Cell Nucleolus , Fibroblast Growth Factor 2 , Fibroblast Growth Factor 2/genetics , Fibroblast Growth Factor 2/pharmacology , Fibroblast Growth Factor 2/metabolism , Cell Nucleolus/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Transcription, Genetic , DNA, Ribosomal/genetics , Chromatin/genetics , Chromatin/metabolismABSTRACT
Physical inactivity is a scourge to human health, promoting metabolic disease and muscle wasting. Interestingly, multiple ecological niches have relaxed investment into physical activity, providing an evolutionary perspective into the effect of adaptive physical inactivity on tissue homeostasis. One such example, the Mexican cavefish Astyanax mexicanus, has lost moderate-to-vigorous activity following cave colonization, reaching basal swim speeds ~3.7-fold slower than their river-dwelling counterpart. This change in behavior is accompanied by a marked shift in body composition, decreasing total muscle mass and increasing fat mass. This shift persisted at the single muscle fiber level via increased lipid and sugar accumulation at the expense of myofibrillar volume. Transcriptomic analysis of laboratory-reared and wild-caught cavefish indicated that this shift is driven by increased expression of pparγ-the master regulator of adipogenesis-with a simultaneous decrease in fast myosin heavy chain expression. Ex vivo and in vivo analysis confirmed that these investment strategies come with a functional trade-off, decreasing cavefish muscle fiber shortening velocity, time to maximal force, and ultimately maximal swimming speed. Despite this, cavefish displayed a striking degree of muscular endurance, reaching maximal swim speeds ~3.5-fold faster than their basal swim speeds. Multi-omic analysis suggested metabolic reprogramming, specifically phosphorylation of Pgm1-Threonine 19, as a key component enhancing cavefish glycogen metabolism and sustained muscle contraction. Collectively, we reveal broad skeletal muscle changes following cave colonization, displaying an adaptive skeletal muscle phenotype reminiscent to mammalian disuse and high-fat models while simultaneously maintaining a unique capacity for sustained muscle contraction via enhanced glycogen metabolism.
Subject(s)
Characidae , Animals , Humans , Characidae/genetics , Biological Evolution , Glycogen , Muscles , Mexico , Caves , MammalsABSTRACT
Paraspeckles are subnuclear RNA-protein structures that are implicated in important processes including cellular stress response, differentiation, and cancer progression. However, it is unclear how paraspeckles impart their physiological effect at the molecular level. Through biochemical analyses, we show that paraspeckles interact with the SWI/SNF chromatin-remodeling complex. This is specifically mediated by the direct interaction of the long-non-coding RNA NEAT1 of the paraspeckles with ARID1B of the cBAF-type SWI/SNF complex. Strikingly, ARID1B depletion, in addition to resulting in loss of interaction with the SWI/SNF complex, decreases the binding of paraspeckle proteins to chromatin modifiers, transcription factors, and histones. Functionally, the loss of ARID1B and NEAT1 influences the transcription and the alternative splicing of a common set of genes. Our findings reveal that dynamic granules such as the paraspeckles may leverage the specificity of epigenetic modifiers to impart their regulatory effect, thus providing a molecular basis for their function.
Subject(s)
Paraspeckles , RNA, Long Noncoding , Transcription Factors/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Chromatin Assembly and Disassembly , Chromatin/geneticsABSTRACT
The nucleolus is sensitive to stress and can orchestrate a chain of cellular events in response to stress signals. Despite being a growth factor, FGF2 has antiproliferative and tumor-suppressive functions in some cellular contexts. In this work, we investigated how the antiproliferative effect of FGF2 modulates chromatin-, nucleolus- and rDNA-associated proteins. The chromatin and nucleolar proteome indicated that FGF2 stimulation modulates proteins related to transcription, rRNA expression and chromatin-remodeling proteins. The global transcriptional rate and nucleolus area increased along with nucleolar disorganization upon 24 h of FGF2 stimulation. FGF2 stimulation induced immature rRNA accumulation by increasing rRNA transcription. The rDNA-associated protein analysis reinforced that FGF2 stimulus interferes with transcription and rRNA processing. RNA Pol I inhibition partially reversed the growth arrest induced by FGF2, indicating that changes in rRNA expression might be crucial for triggering the antiproliferative effect. Taken together, we demonstrate that the antiproliferative FGF2 stimulus triggers significant transcriptional changes and modulates the main cell transcription site, the nucleolus.
ABSTRACT
The nucleolus is sensitive to stress and can orchestrate a chain of cellular events in response to stress signals. Despite being a growth factor, FGF2 has antiproliferative and tumor-suppressive functions in some cellular contexts. In this work, we investigated how the antiproliferative effect of FGF2 modulates chromatin-, nucleolus-, and rDNA-associated proteins. The chromatin and nucleolar proteome indicated that FGF2 stimulation modulates proteins related to transcription, rRNA expression, and chromatin remodeling proteins. The global transcriptional rate and nucleolus area increased along with nucleolar disorganization upon 24 h of FGF2 stimulation. FGF2 stimulation induced immature rRNA accumulation by increasing rRNA transcription. The rDNA-associated protein analysis reinforced that FGF2 stimulus interferes with transcription and rRNA processing. RNA Pol I inhibition partially reversed the growth arrest induced by FGF2, indicating that changes in rRNA expression may be crucial for triggering the antiproliferative effect. Taken together, we demonstrate that the antiproliferative FGF2 stimulus triggers significant transcriptional changes and modulates the main cell transcription site, the nucleolus.
ABSTRACT
Synthetic messenger RNA (mRNA) is an emerging therapeutic platform with important applications in oncology and infectious disease. Effective mRNA medicines must be translated by the ribosome but not trigger a strong nucleic acid-mediated immune response. To expand the medicinal chemistry toolbox for these agents, here we report the properties of the naturally occurring nucleobase N4-acetylcytidine (ac4C) in synthetic mRNAs. We find that ac4C is compatible with, but does not enhance, protein production in the context of synthetic mRNA reporters. However, replacement of cytidine with ac4C diminishes inflammatory gene expression in immune cells caused by synthetic mRNAs. Chemoproteomic capture indicates that ac4C alters the protein interactome of synthetic mRNAs, reducing binding to cytidine-binding proteins and an immune sensor. Overall, our studies illustrate the unique ability of ac4C to modulate RNA-protein interactions and provide a foundation for using N4-cytidine acylation to fine-tune the properties of nucleic acid therapeutics.
Subject(s)
Cytidine/metabolism , Inflammation/metabolism , RNA, Messenger/metabolism , Acetylation , Cells, Cultured , Humans , Protein Processing, Post-TranslationalABSTRACT
Telomerase reverse transcriptase (TERT) and the noncoding telomerase RNA (TR) subunit constitute the core of telomerase. Additional subunits are required for ribonucleoprotein complex assembly and in some cases remain stably associated with the active holoenzyme. Pof8, a member of the LARP7 protein family is such a constitutive component of telomerase in fission yeast. Using affinity purification of Pof8, we have identified two previously uncharacterized proteins that form a complex with Pof8 and participate in telomerase biogenesis. Both proteins participate in ribonucleoprotein complex assembly and are required for wildtype telomerase activity and telomere length maintenance. One factor we named Thc1 (Telomerase Holoenzyme Component 1) shares structural similarity with the nuclear cap binding complex and the poly-adenosine ribonuclease (PARN), the other is the ortholog of the methyl phosphate capping enzyme (Bin3/MePCE) in metazoans and was named Bmc1 (Bin3/MePCE 1) to reflect its evolutionary roots. Thc1 and Bmc1 function together with Pof8 in recognizing correctly folded telomerase RNA and promoting the recruitment of the Lsm2-8 complex and the catalytic subunit to assemble functional telomerase.
Subject(s)
Schizosaccharomyces , Telomerase , Holoenzymes/metabolism , Phosphates/metabolism , Protein Binding , RNA/metabolism , RNA Cap-Binding Proteins/metabolism , Schizosaccharomyces/metabolism , Telomerase/metabolism , Telomere/metabolismABSTRACT
SETD2 is an important methyltransferase that methylates crucial substrates such as histone H3, tubulin, and STAT1 and also physically interacts with transcription and splicing regulators such as Pol II and various hnRNPs. Of note, SETD2 has a functionally uncharacterized extended N-terminal region, the removal of which leads to its stabilization. How this region regulates SETD2 half-life is unclear. Here we show that SETD2 consists of multiple long disordered regions across its length that cumulatively destabilize the protein by facilitating its proteasomal degradation. SETD2 disordered regions can reduce the half-life of the yeast homolog Set2 in mammalian cells as well as in yeast, demonstrating the importance of intrinsic structural features in regulating protein half-life. In addition to the shortened half-life, by performing fluorescence recovery after photobleaching assay we found that SETD2 forms liquid droplets in vivo, another property associated with proteins that contain disordered regions. The phase-separation behavior of SETD2 is exacerbated upon the removal of its N-terminal segment and results in activator-independent histone H3K36 methylation. Our findings reveal that disordered region-facilitated proteolysis is an important mechanism governing SETD2 function.
Subject(s)
Histone-Lysine N-Methyltransferase/physiology , Intrinsically Disordered Proteins/physiology , Fluorescence Recovery After Photobleaching/methods , HEK293 Cells , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Humans , Intrinsically Disordered Proteins/metabolism , Mass Spectrometry/methods , Methylation , Methyltransferases/metabolism , Methyltransferases/physiology , Protein Binding , Protein Processing, Post-Translational , Protein Stability , Proteolysis , Structure-Activity RelationshipABSTRACT
The extracellular matrix (ECM) is a three-dimensional network of macromolecules that provides a microenvironment capable of supporting and regulating cell functions. However, only a few research organisms are available for the systematic dissection of the composition and functions of the ECM, particularly during regeneration. We utilized the free-living flatworm Schmidtea mediterranea to develop an integrative approach consisting of decellularization, proteomics, and RNAi to characterize and investigate ECM functions during tissue homeostasis and regeneration. ECM-enriched samples were isolated from planarians, and their proteomes were characterized by LC-MS/MS. The functions of identified ECM components were interrogated using RNA interference. Using this approach, we found that heparan sulfate proteoglycan is essential for tissue regeneration. Our strategy provides an experimental approach for identifying both known and novel ECM components involved in regeneration.
Subject(s)
Decellularized Extracellular Matrix , Planarians , Regeneration , Animals , Helminth Proteins/genetics , Helminth Proteins/metabolism , Heparan Sulfate Proteoglycans , Homeostasis , Planarians/genetics , Planarians/metabolism , Planarians/physiology , Proteome , RNA InterferenceABSTRACT
Heterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.
Subject(s)
Alternative Splicing/genetics , Heterogeneous-Nuclear Ribonucleoprotein L/metabolism , Histone-Lysine N-Methyltransferase/metabolism , RNA Precursors/genetics , RNA, Messenger/genetics , Cell Line , HEK293 Cells , Histone-Lysine N-Methyltransferase/genetics , Histones/metabolism , Humans , Protein Domains/physiology , RNA Polymerase II/metabolismABSTRACT
Chemical proteomics provides a powerful strategy for the high-throughput assignment of enzyme function or inhibitor selectivity. However, identifying optimized probes for an enzyme family member of interest and differentiating signal from the background remain persistent challenges in the field. To address this obstacle, here we report a physiochemical discernment strategy for optimizing chemical proteomics based on the coenzyme A (CoA) cofactor. First, we synthesize a pair of CoA-based sepharose pulldown resins differentiated by a single negatively charged residue and find this change alters their capture properties in gel-based profiling experiments. Next, we integrate these probes with quantitative proteomics and benchmark analysis of "probe selectivity" versus traditional "competitive chemical proteomics." This reveals that the former is well-suited for the identification of optimized pulldown probes for specific enzyme family members, while the latter may have advantages in discovery applications. Finally, we apply our anionic CoA pulldown probe to evaluate the selectivity of a recently reported small molecule N-terminal acetyltransferase inhibitor. These studies further validate the use of physical discriminant strategies in chemoproteomic hit identification and demonstrate how CoA-based chemoproteomic probes can be used to evaluate the selectivity of small molecule protein acetyltransferase inhibitors, an emerging class of preclinical therapeutic agents.
Subject(s)
Acetyltransferases/chemistry , Molecular Probes/chemistry , Proteomics/methods , Amino Acid Sequence , Chromatography, High Pressure Liquid/methods , Electrophoresis, Polyacrylamide Gel/methods , Reproducibility of Results , Substrate Specificity , Tandem Mass Spectrometry/methodsABSTRACT
N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA1-3. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. In human and yeast mRNAs, ac4C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease4-6.
Subject(s)
Acetylation , Cytidine/analogs & derivatives , Eukaryotic Cells/metabolism , Evolution, Molecular , RNA/chemistry , RNA/metabolism , Archaea/chemistry , Archaea/cytology , Archaea/genetics , Archaea/growth & development , Conserved Sequence , Cryoelectron Microscopy , Cytidine/metabolism , Eukaryotic Cells/cytology , HeLa Cells , Humans , Models, Molecular , N-Terminal Acetyltransferases/metabolism , RNA, Archaeal/chemistry , RNA, Archaeal/genetics , RNA-Binding Proteins/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Ribosomes/ultrastructure , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Sequence Analysis, DNA , TemperatureABSTRACT
Acyl-coenzyme A (CoA)/protein interactions are essential for life. Despite this importance, their global scope and selectivity remains undefined. Here, we describe CATNIP (CoA/AcetylTraNsferase Interaction Profiling), a chemoproteomic platform for the high-throughput analysis of acyl-CoA/protein interactions in endogenous proteomes. First, we apply CATNIP to identify acetyl-CoA-binding proteins through unbiased clustering of competitive dose-response data. Next, we use this method to profile the selectivity of acyl-CoA/protein interactions, leading to the identification of specific acyl-CoA engagement signatures. Finally, we apply systems-level analyses to assess the features of protein networks that may interact with acyl-CoAs, and use a strategy for high-confidence proteomic annotation of acetyl-CoA-binding proteins to identify a site of non-enzymatic acylation in the NAT10 acetyltransferase domain that is likely driven by acyl-CoA binding. Overall, our studies illustrate how chemoproteomics and systems biology can be integrated to understand the roles of acyl-CoA metabolism in biology and disease.
Subject(s)
Acyl Coenzyme A/chemistry , Protein Interaction Maps , Proteins/chemistry , Proteomics , Acyl Coenzyme A/metabolism , Humans , Proteins/metabolismABSTRACT
The kinetochore is a large molecular machine that attaches chromosomes to microtubules and facilitates chromosome segregation. The kinetochore includes submodules that associate with the centromeric DNA and submodules that attach to microtubules. Additional copies of several submodules of the kinetochore are added during anaphase, including the microtubule binding module Ndc80. While the factors governing plasticity are not known, they could include regulation based on microtubule-kinetochore interactions. We report that Fin1 localizes to the microtubule-proximal edge of the kinetochore cluster during anaphase based on single-particle averaging of super-resolution images. Fin1 is required for the assembly of normal levels of Dam1 and Ndc80 submodules. Levels of Ndc80 further depend on the Dam1 microtubule binding complex. Our results suggest the stoichiometry of outer kinetochore submodules is strongly influenced by factors at the kinetochore-microtubule interface such as Fin1 and Dam1, and phosphorylation by cyclin-dependent kinase. Outer kinetochore stoichiometry is remarkably plastic and responsive to microtubule-proximal regulation.
Subject(s)
Cell Cycle Proteins/genetics , Chromosome Segregation/genetics , Cytoskeletal Proteins/genetics , Microtubule-Associated Proteins/genetics , Nuclear Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Anaphase/genetics , Centromere/genetics , Chromosomes/genetics , Cyclin-Dependent Kinases/genetics , Kinetochores/metabolism , Microtubules/genetics , Phosphorylation/genetics , Protein Binding/genetics , Saccharomyces cerevisiae/geneticsABSTRACT
Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth. Understanding these mechanisms thus represents a vital goal. Toward this goal, here we report a chemoproteomic map of fumarate, a covalent oncometabolite whose accumulation marks the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). We applied a fumarate-competitive chemoproteomic probe in concert with LC-MS/MS to discover new cysteines sensitive to fumarate hydratase (FH) mutation in HLRCC cell models. Analysis of this dataset revealed an unexpected influence of local environment and pH on fumarate reactivity, and enabled the characterization of a novel FH-regulated cysteine residue that lies at a key protein-protein interface in the SWI-SNF tumor-suppressor complex. Our studies provide a powerful resource for understanding the covalent imprint of fumarate on the proteome and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.
Subject(s)
Fumarates/metabolism , Leiomyomatosis/metabolism , Neoplastic Syndromes, Hereditary/metabolism , Skin Neoplasms/metabolism , Uterine Neoplasms/metabolism , Cell Line, Tumor , Chromatography, Liquid/methods , Cysteine , HEK293 Cells , Humans , Hydrogen-Ion Concentration , Leiomyomatosis/genetics , Models, Biological , Neoplastic Syndromes, Hereditary/genetics , Proteomics , Signal Transduction , Skin Neoplasms/genetics , Tandem Mass Spectrometry/methods , Uterine Neoplasms/geneticsABSTRACT
The use of mass spectrometry as a tool to detect proteins of biological interest has become a cornerstone of proteomics. The popularity of mass spectrometry-based methods has increased along with instrument improvements in detection and speed. The Orbitrap Fusion Lumos mass spectrometer has recently been shown to have better fragmentation and detection than its predecessors. Here, we determined the sensitivity of the Lumos using the NIST monoclonal antibody reference material at various concentrations to detect its peptides in a background of S. cerevisiae whole cell lysate, which was kept at a constant concentration. The data collected by data-dependent acquisition showed that the spiked protein could be detected at 10 pg by an average of 4 peptides in 250 ng of whole cell lysate when the instrument was operated by detecting the peptide masses in the Orbitrap and the fragment masses in the ion trap (FTIT mode). In contrast, when the peptides and fragments were both detected in the Orbitrap on either the Lumos or Q-Exactive Plus (FTFT mode), the lowest concentration of NIST monoclonal antibody detected was 50 pg. The Lumos can detect a single protein at a level 2500 times lower than the whole cell background and the combination of detecting ions in the Orbitrap and ion trap can improve the identification of low abundance proteins. Furthermore, the total number of proteins identified from decreasing starting amounts of whole cell extracts was determined. The Lumos, when operated in FTIT mode, was able to identify twice as many proteins compared to the Q-Exactive+ at 5 ng of whole cell lysate. Similar numbers of proteins were identified on both platforms at higher concentrations of starting material. Therefore, the Lumos mass spectrometer is especially useful for detecting proteins of low abundance in complex backgrounds or samples that have limited starting material.
Subject(s)
Mass Spectrometry/methods , Peptides/metabolism , Proteins/metabolism , Humans , Ions/metabolism , Peptide Fragments/metabolism , Proteomics/methods , Reproducibility of Results , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Eukaryotic gene regulation is a complex process, often coordinated by the action of tens to hundreds of proteins. Although previous biochemical studies have identified many components of the basal machinery and various ancillary factors involved in gene regulation, numerous gene-specific regulators remain undiscovered. To comprehensively survey the proteome directing gene expression at a specific genomic locus of interest, we developed an in vitro nuclease-deficient Cas9 (dCas9)-targeted chromatin-based purification strategy, called "CLASP" (Cas9 locus-associated proteome), to identify and functionally test associated gene-regulatory factors. Our CLASP method, coupled to mass spectrometry and functional screens, can be efficiently adapted for isolating associated regulatory factors in an unbiased manner targeting multiple genomic loci across different cell types. Here, we applied our method to isolate the Drosophila melanogaster histone cluster in S2 cells to identify several factors including Vig and Vig2, two proteins that bind and regulate core histone H2A and H3 mRNA via interaction with their 3' UTRs.
Subject(s)
Bacterial Proteins/genetics , Chromatin/isolation & purification , Chromosomal Proteins, Non-Histone/genetics , Drosophila Proteins/genetics , Endonucleases/genetics , Genes, Regulator/genetics , Histones/genetics , RNA-Binding Proteins/genetics , 3' Untranslated Regions , Animals , Bacterial Proteins/metabolism , CRISPR-Associated Protein 9 , Chromatin/genetics , Chromatin Immunoprecipitation , Chromosomal Proteins, Non-Histone/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Endonucleases/metabolism , Gene Expression , Histones/metabolism , Humans , Promoter Regions, Genetic , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism , RNA-Binding Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Monoclonal antibody (mAb) drugs constitute the largest class of protein therapeutics currently on the market. Correctly folded protein higher order structure (HOS), including quinary structure, is crucial for mAb drug quality. The quinary structure is defined as the association of quaternary structures (e.g., oligomerized mAb). Here, several commonly available analytical methods, i.e., size-exclusion-chromatography (SEC) FPLC, multi-angle light scattering (MALS), circular dichroism (CD), NMR and multivariate analysis, were combined and modified to yield a complete profile of HOS and comparable metrics. Rituximab and infliximab were chosen for method evaluation because both IgG1 molecules are known to be homologous in sequence, superimposable in Fab crystal structure and identical in Fc structure. However, herein the two are identified to be significantly different in quinary structure in addition to minor secondary structure differences. All data collectively showed rituximab was mostly monomeric while infliximab was in mono-oligomer equilibrium driven by its Fab fragment. The quinary structure differences were qualitatively inferred from the less used but more reproducible dilution-injection-SEC-FPLC curve method. Quantitative principal component analysis (PCA) was performed on NMR spectra of either the intact or the in-situ enzymatic-digested mAb samples. The cleavage reactions happened directly in NMR tubes without further separation, which greatly enhanced NMR spectra quality and resulted in larger inter- and intra-lot variations based on PCA. The new in-situ enzymatic digestion method holds potential in identifying structural differences on larger therapeutic molecules using NMR.
Subject(s)
Infliximab/chemistry , Magnetic Resonance Spectroscopy/methods , Protein Conformation , Rituximab/chemistry , Chemistry Techniques, Analytical , Immunoglobulin Fab Fragments/chemistryABSTRACT
Conjugated estrogens purified from pregnant mares urine has been used as estrogen hormone replacement therapy since 1942. Previously, methods were proposed to identify and quantify the components of this complex mixture but ultimately were withdrawn due to incomplete characterization of the product and difficulties in transferring the method between laboratories. The aim of the current study is to develop a LC method that can reliably detect multiple steroidal components in conjugated estrogen tablets and measure their relative amount. The method developed was optimized for UHPLC columns, and the elution profile was analyzed using high-resolution mass spectrometry. A total of 60 steroidal components were identified using their exact m/z, product ion spectra of known, and predicted conjugated estrogen structures. These components were consistently present in 23 lots of Premarin tablets spanning two production years. The ten conjugated estrogens identified in the USP monograph and other additional estrogens reported elsewhere are among the 60 steroidal components reported here. The LC-MS method was tested in different laboratories using multiple samples, and the obtained results were reproducible among laboratories.
Subject(s)
Drug Contamination , Estrogens, Conjugated (USP)/analysis , Estrogens, Conjugated (USP)/chemistry , Mass Spectrometry/methods , Animals , Chromatography, Liquid/methods , Female , Horses , PregnancyABSTRACT
Mass spectrometry has gained widespread acceptance for the characterization of protein therapeutics as a part of the regulatory approval process. Improvements in mass spectrometer sensitivity, resolution, and mass accuracy have enabled more detailed and confident analysis of larger biomolecules for confirming amino acid sequences, assessing sequence variants, and characterizing post translational modifications. This work demonstrates the suitability of a combined approach using intact MS and multistage top down MS/MS analyses for the characterization of a protein therapeutic drug. The protein therapeutic granulocyte-colony stimulating factor was analyzed using a Thermo Fusion Tribrid mass spectrometer using a multistage top down MS approach. Intact mass analysis identified the presence of two disulfide bonds based on exact mass shifts while a combined collision induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) MS/MS approach obtained 80% protein sequence coverage. Isolating MS/MS fragments for MS(3) analysis using HCD or CID increased the sequence coverage to 89%. 95% sequence coverage was obtained by reducing human granulocyte-colony stimulating factor (G-CSF) prior to MS/MS and MS(3) analysis to specifically target the residues between the disulfide bonds. The use of this combined intact MS and multistage top down MS approach allows for rapid and accurate determination of the primary sequence of a protein therapeutic drug product.