RESUMO
The chemical characterization of extractables and leachables (E&Ls) is an important aspect of biosafety and biocompatibility assessment in medical device industry. The advent of the body-contact use of medical devices in patient treatment has introduced a potential source for extractables and leachables as these medical devices are comprised of various polymeric materials. Several industry working groups, the FDA and USP, have recognized the guidance for chemical characterizations and nontargeted analysis of medical device extracts, such as ISO 10993-18:2020. The MS application of nontargeted analysis has played a critical role in understanding the E&Ls from medical device extracts. However, there have been very few reports about the MS based workflow with nontargeted analysis for medical device extracts and there is little guidance about the exact methodologies which should be used, even though there is an urgent need for a clearly defined process for the identification of medical device extracts. In this study, we demonstrated an analytical LC/MS (liquid chromatography/mass spectrometry) workflow using high resolution Exploris120 Orbitrap instrument for data acquisition and Compound Discoverer 3.3 intelligent software for data processing to profile the polymer related E&Ls from a balloon dilation catheter device extracted with 40% ethanol. An E&L ID workflow combining LC separation, data-informed MS acquisition strategy, MS information mining (including adduct ions, MS information from both electrospray ionization (ESI) (+) and ESI (-), in-source fragmentation, common fragment ions (CFIs), common neutral losses (CNLs), and in silico MS simulation was described with intelligent software processing and manual data interpretation. The workflow developed in this study was proven to be effective to provide a comprehensive profile of polymer related degradation products, polymer impurities and additives including surfactants, UV curing agent, antioxidants, and plasticizers for the device analyzed. The classification of E&L compounds using CFIs and CNLs was very effective to facilitate the identification of polymer related impurities and extract the polymer related impurities with common structures in a large data result set.
Assuntos
Misturas Complexas , Polímeros , Humanos , Fluxo de Trabalho , Espectrometria de Massas , Cromatografia LíquidaRESUMO
Reduced brain metabolism is an invariant feature of Alzheimer Disease (AD) that is highly correlated to the decline in brain functions. Decreased activities of key tricarboxylic acid cycle (TCA) cycle enzymes may underlie this abnormality and are highly correlated to the clinical state of the patient. The activity of the α-ketoglutarate dehydrogenase complex (KGDHC), an arguably rate-limiting enzyme of the TCA cycle, declines with AD, but the mechanism of inactivation and whether it can be reversed remains unknown. KGDHC consists of multiple copies of three subunits. KGDHC is sensitive to oxidative stress, which is pervasive in AD brain. The present studies tested the mechanism for the peroxynitrite-induced inactivation and subsequent reactivation of purified and cellular KGDHC. Peroxynitrite inhibited purified KGDHC activity in a dose-dependent manner and reduced subunit immunoreactivity and increased nitrotyrosine immunoreactivity. Nano-LC-MS/MS showed that the inactivation was related to nitration of specific tyrosine residues in the three subunits. GSH diminished the nitrotyrosine immunoreactivity of peroxynitrite-treated KGDHC, restored the activity and the immunoreactivity for KGDHC. Nano-LC-MS/MS showed this was related to de-nitration of specific tyrosine residues, suggesting KGDHC may have a denitrase activity. Treatment of N2a cells with peroxynitrite for 5 min followed by recovery of cells for 24 h reduced KGDHC activity and increased nitrotyrosine immunoreactivity. Increasing cellular GSH in peroxynitrite-treated cells rescued KGDHC activity to the control level. The results suggest that restoring KGDHC activity is possible and may be a useful therapeutic approach in neurodegenerative diseases.
Assuntos
Complexo Cetoglutarato Desidrogenase/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Ácido Peroxinitroso/farmacologia , Tirosina/análogos & derivados , Doença de Alzheimer/enzimologia , Doença de Alzheimer/terapia , Encéfalo/enzimologia , Linhagem Celular , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Complexo Cetoglutarato Desidrogenase/química , Proteínas Mitocondriais/química , Ácido Peroxinitroso/química , Ácido Peroxinitroso/metabolismo , Tirosina/química , Tirosina/metabolismo , Tirosina/farmacologiaRESUMO
The snowboarding project has the characteristics of high risk and high technical level. The current publicity level is not high, and the number of participants is also very limited. Another potential advantage medal breakthrough project that is expected to be achieved in the Winter Olympics has received a lot of attention, creating favorable opportunities for the promotion and development of this project in China. The event requires good special physical support, skeletal muscle contraction is the body to produce motor function, and special physical training and recovery are key factors for athletes to obtain excellent results in the competition. This article is aimed at performing ultrasonic quantitative analysis on the skeletal muscles of skiers after exercise based on artificial intelligence and complex networks and at studying the skeletal muscle conditions of snowboarders after exercise, so as to provide a certain theoretical basis for coaches in future scientific training. Based on a large amount of literature, this paper uses variational optical flow calculation and split Bregman method to solve the typical HS model, L1-L2 model, and L1-high-order model, respectively, and uses the motion estimation method to describe the movement of muscles. An experiment was designed to collect ultrasound images of the gastrocnemius and quadriceps muscles during contraction. In addition, a motion target positioning algorithm was used to obtain some motion parameters, which provided direct help for athletes in rationally arranging training load and scientific training. The experimental results in this paper show that the muscle motion features extracted from the ultrasound sequence images can quantitatively express a lot of important information about the skeletal muscle motion form and function and have potential practical application value. And the different invariants of each type of ski trajectory vary greatly, floating between 1.5429 and 7.6759.
RESUMO
Titanium alloy is a typical difficult-to-machine material with features of superhigh strength and hardness, and low elastic modulus. It is difficult to guarantee the processing quality and efficiency due to the high cutting force and tool wear in conventional cutting. Elliptical vibration cutting (EVC) as an effective method can improve the machinability of titanium alloys. In this paper, the finite element method (FEM) was adopted to study the cutting force and residual stress of 3D EVC in machining of Ti6Al4V. The Johnson-Cook constitutive model was utilized to illustrate the plastic behavior of Ti6Al4V alloy. The kinematics of the 3D EVC was described, and then the influence of various cutting speeds, vibration amplitudes, vibration frequencies and depths of cut on cutting force and residual stress were carried out and analyzed. The simulation results show that the cutting speed, vibration amplitude a, vibration frequency and depth of cut have larger effect on principal force. In addition, the compressive stress layer can be easily obtained near the machined surface by using 3D EVC, which is helpful to improve the working performance of workpiece.
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Extractables or leachables of biomaterials or residues of additives used in the manufacturing process that are potentially released from a medical device may have an adverse effect on a patient. Chemical characterization of leachable chemicals and degradation products in a medical device is an important aspect of its overall biocompatibility assessment process, which helps to ensure that the therapeutical benefits exceed the potential biological risks associated with the use of the device or its components or materials. By evaluating the types and amounts of chemicals that may migrate from a device to a patient during clinical use, potential toxicological risks can be assessed. A semipolar solvent, 40% ethanol in water (v/v), an appropriate surrogate for blood and blood related substances, was used as an extraction medium to mimic the body fluid in contact with a medical device. The extraction was conducted at 37 °C for 24 h for limited exposure medical devices per ISO 10993-12:2021. From gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) analysis, leachable chemicals of polylactams, linear polyamides, cyclic polytetramethylene ether (PTME), poly(tetramethylene ether) glycol (PTMEG), cyclic and linear poly(tetramethylene ether) glycol adipate (PTMEGA), cyclic and linear poly(tetramethylene ether) glycol adipamide (PTMEGAA) were structurally elucidated. The workflow presented in this study was proven to be a successful approach for rapid extractable and leachable profiling and identification with confidence.
RESUMO
Major histocompatibility complex class II (MHC II) molecules are expressed on the surface of antigen-presenting cells and display short bound peptide fragments derived from self- and nonself antigens. These peptide-MHC complexes function to maintain immunological tolerance in the case of self-antigens and initiate the CD4(+) T cell response in the case of foreign proteins. Here we report the application of LC-MS/MS analysis to identify MHC II peptides derived from endogenous proteins expressed in freshly isolated murine splenic DCs. The cell number was enriched in vivo upon treatment with Flt3L-B16 melanoma cells. In a typical experiment, starting with about 5 × 10(8) splenic DCs, we were able to reliably identify a repertoire of over 100 MHC II peptides originating from about 55 proteins localized in membrane (23%), intracellular (26%), endolysosomal (12%), nuclear (14%), and extracellular (25%) compartments. Using synthetic isotopically labeled peptides corresponding to the sequences of representative bound MHC II peptides, we quantified by LC-MS relative peptide abundance. In a single experiment, peptides were detected in a wide concentration range spanning from 2.5 fmol/µL to 12 pmol/µL or from approximately 13 to 2 × 10(5) copies per DC. These peptides were found in similar amounts on B cells where we detected about 80 peptides originating from 55 proteins distributed homogenously within the same cellular compartments as in DCs. About 90 different binding motifs predicted by the epitope prediction algorithm were found within the sequences of the identified MHC II peptides. These results set a foundation for future studies to quantitatively investigate the MHC II repertoire on DCs generated under different immunization conditions.
Assuntos
Apresentação de Antígeno , Células Dendríticas/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Fragmentos de Peptídeos/metabolismo , Baço/citologia , Algoritmos , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Transplante de Neoplasias , Fragmentos de Peptídeos/química , Cultura Primária de Células , Baço/imunologia , Espectrometria de Massas em TandemRESUMO
Elucidating physiological and pathogenic functions of protein methyltransferases (PMTs) relies on knowing their substrate profiles. S-adenosyl-L-methionine (SAM) is the sole methyl-donor cofactor of PMTs. Recently, SAM analogues have emerged as novel small-molecule tools to efficiently label PMT substrates. Here we reported the development of a clickable SAM analogue cofactor, 4-propargyloxy-but-2-enyl SAM, and its implementation to label substrates of human protein arginine methyltransferase 1 (PRMT1). In the system, the SAM analogue cofactor, coupled with matched PRMT1 mutants rather than native PRMT1, was shown to label PRMT1 substrates. The transferable 4-propargyloxy-but-2-enyl moiety of the SAM analogue further allowed corresponding modified substrates to be characterized through a subsequent click chemical ligation with an azido-based probe. The SAM analogue, in combination with a rational protein-engineering approach, thus shows potential to label and identify PMT targets in the context of a complex cellular mixture.
Assuntos
Proteína-Arginina N-Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Engenharia de Proteínas , Proteína-Arginina N-Metiltransferases/química , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Bone remodeling relies on a dynamic balance between bone formation and resorption, mediated by osteoblasts and osteoclasts, respectively. Under certain stimuli, osteoprogenitor cells may differentiate into premature osteoblasts and further into mature osteoblasts. This process is marked by increased alkaline phosphatase (ALP) activity and mineralized nodule formation. In this study, we induced osteoblast differentiation in mouse osteoprogenitor MC3T3-E1 cells and divided the process into three stages. In the first stage (day 3), the MC3T3-E1 cell under osteoblast differentiation did not express ALP or deposit a mineralized nodule. In the second stage, the MC3T3-E1 cell expressed ALP but did not form a mineralized nodule. In the third stage, the MC3T3-E1 cell had ALP activity and formed mineralized nodules. In the present study, we focused on morphological and proteomic changes of MC3T3-E1 cells in the early stage of osteoblast differentiation - a period when premature osteoblasts transform into mature osteoblasts. We found that mean cell area and mean stress fiber density were increased in this stage due to enhanced cell spreading and decreased cell proliferation. We further analyzed the proteins in the signaling pathway of regulation of the cytoskeleton using a proteomic approach and found upregulation of IQGAP1, gelsolin, moesin, radixin, and Cfl1. After analyzing the focal adhesion signaling pathway, we found the upregulation of FLNA, LAMA1, LAMA5, COL1A1, COL3A1, COL4A6, and COL5A2 as well as the downregulation of COL4A1, COL4A2, and COL4A4. In conclusion, the signaling pathway of regulation of the cytoskeleton and focal adhesion play critical roles in regulating cell spreading and actin skeleton formation in the early stage of osteoblast differentiation.
Assuntos
Diferenciação Celular , Osteoblastos/citologia , Proteômica , Células-Tronco/citologia , Actinas/metabolismo , Animais , Western Blotting , Adesão Celular , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas , Camundongos , Transdução de SinaisRESUMO
Advances in immunology and immune therapies require knowledge of antigenic peptide sequences that are presented on MHC class II and class I molecules of antigen presenting cells. The most specialized antigen presenting cells are dendritic cells (DCs). In the past, the small number of DCs that could be isolated from mouse spleen prevented direct analysis of the MHC II peptide repertoire presented by DCs. Here we describe a protocol that integrates immunological methods (in vivo enrichment of mouse spleen DCs by Flt3L treatment and immunoprecipitation of MHC II-peptide complexes), mass spectrometry analysis and peptide synthesis (LC-MS/MS and quantitation analysis for non tryptic peptides) to identify and quantitate the endogenous peptides that are bound to MHC II molecules on DCs. The described method produces quantitative data that are reproducible and reliable enough to cover a wide range of peptide copy numbers. We propose the application of this method in future studies to quantitatively investigate the MHC II repertoire on DCs presented during viral infections or different immunizations in vaccine development research.
Assuntos
Células Dendríticas/metabolismo , Genes MHC da Classe II/fisiologia , Imunoprecipitação/métodos , Peptídeos/análise , Peptídeos/metabolismo , Baço/citologia , Espectrometria de Massas em Tandem/métodos , Animais , CamundongosRESUMO
Mass Spectrometry (MS) is becoming a preferred method to identify class I and class II peptides presented on major histocompability complexes (MHC) on antigen presenting cells (APC). We describe a combined computational and MS approach to identify exogenous MHC II peptides presented on mouse spleen dendritic cells (DCs). This approach enables rapid, effective screening of a large number of possible peptides by a computer-assisted strategy that utilizes the extraordinary human ability for pattern recognition. To test the efficacy of the approach, a mixture of epitope peptide mimics (mimetopes) from HIV gag p24 sequence were added exogenously to Fms-like tyrosine kinase 3 ligand (Flt3L)-mobilized splenic DCs. We identified the exogenously added peptide, VDRFYKTLRAEQASQ, and a second peptide, DRFYKLTRAEQASQ, derived from the original exogenously added 15-mer peptide. Furthermore, we demonstrated that our strategy works efficiently with HIV gag p24 protein when delivered, as vaccine protein, to Flt3L expanded mouse splenic DCs in vitro through the DEC-205 receptor. We found that the same MHC II-bound HIV gag p24 peptides, VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ, were naturally processed from anti-DEC-205 HIV gag p24 protein and presented on DCs. The two identified VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ MHC II-bound HIV gag p24 peptides elicited CD4(+) T-cell mediated responses in vitro. Their presentation by DCs to antigen-specific T cells was inhibited by chloroquine (CQ), indicating that optimal presentation of these exogenously added peptides required uptake and vesicular trafficking in mature DCs. These results support the application of our strategy to identify and characterize peptide epitopes derived from vaccine proteins processed by DCs and thus has the potential to greatly accelerate DC-based vaccine development.
Assuntos
Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/metabolismo , Epitopos de Linfócito T/imunologia , Proteína do Núcleo p24 do HIV/imunologia , Sequência de Aminoácidos , Animais , Apresentação de Antígeno , Antígenos Virais/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Células Dendríticas/imunologia , Epitopos de Linfócito T/metabolismo , Proteína do Núcleo p24 do HIV/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismoRESUMO
Protein lysine methyltransferases (PKMTs) play crucial roles in normal physiology and disease processes. Profiling PKMT targets is an important but challenging task. With cancer-relevant G9a as a target, we have demonstrated success in developing S-adenosyl-L-methionine (SAM) analogues, particularly (E)-hex-2-en-5-ynyl SAM (Hey-SAM), as cofactors for engineered G9a. Hey-SAM analogue in combination with G9a Y1154A mutant modifies the same set of substrates as their native counterparts with remarkable efficiency. (E)-Hex-2-en-5-ynylated substrates undergo smooth click reaction with an azide-based probe. This approach is thus suitable for substrate characterization of G9a and expected to further serve as a starting point to evolve other PKMTs to utilize a similar set of cofactors.
Assuntos
Coenzimas/química , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , S-Adenosilmetionina/análogos & derivados , Coenzimas/metabolismo , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Humanos , Mutação , S-Adenosilmetionina/metabolismoRESUMO
Mass spectrometric profiling using ProteinChip and magnetic beads has rapidly grown over the past years, particularly to generate serum profiles for cancer diagnosis. The molecular weights of these distinguishing peaks are usually under 30 kDa. To identify those low molecular weight proteins and peptides is important for specific assays to be developed and increases biological insight. In this study, low molecular weight proteins and peptides from serum were purified by a combination of weak cation exchange magnetic beads and high performance liquid chromatography. The purified proteins and peptides were analyzed by 1D SDS PAGE, SELDI and LC-MS/MS. 246 proteins were identified from the HPLC fractions by LC-MS/MS. 95(38.62%) proteins were first identified in serum compare with Sys-BodyFluid database. 11(11/96) proteins were documented cancer associated proteins. We also observed about 109 proteins/peptides in SELDI mass spectrum, and 13 of the SELDI features were identified.
Assuntos
Proteínas Sanguíneas/análise , Peptídeos/sangue , Proteômica/métodos , Proteínas Sanguíneas/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas , Peso Molecular , Análise Serial de ProteínasRESUMO
The disulfide bonds in the galactose-specific lectin SEL 24K from the egg of the Chinook salmon Oncorhynchus tshawytscha were determined by mass spectrometry. Four predictive in silico tools were used to determine the oxidation state of cysteines in the sequence and possible location of the disulfide bonds. A combination of tryptic digestion, HPLC separation, and chemical modifications were used to establish the location of seven disulfide bonds and one pair of free cysteines. After proteolysis, peptides containing one or two disulfide bonds were identified by reduction and mass spectral comparison. MALDI mass spectrometry was supported by chemical modification (iodoacetamide) and in silico digestion. The assignments of disulfide bonds were further confirmed by mass spectral fragmentation studies including in-source dissociation (ISD) and collision-induced dissociation (CID). The experimentally determined disulfide bonds and free Cys residues were only partially consistent with those generated by several automated public-domain algorithms.
Assuntos
Dissulfetos/química , Proteínas do Ovo/química , Proteínas de Peixes/química , Lectinas/química , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Cistina/análise , Proteínas do Ovo/isolamento & purificação , Proteínas de Peixes/isolamento & purificação , Lectinas/isolamento & purificação , Dados de Sequência Molecular , Oxirredução , Fragmentos de Peptídeos/isolamento & purificação , Salmão , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/metabolismoRESUMO
Quantitative analysis of free fatty acids was achieved using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with a meso-tetrakis porphyrin matrix. Cesium acetate was employed as a cationizing agent. The MALDI signal was reproducible and dominated by cesiated cesium carboxylates [RCOOCs + Cs]+. The addition of two Cs ions resulted in a mass shift of 264.8 Da for each fatty acid and greatly reduced background peaks. A linear relationship between fatty acid concentration and corresponding fatty acid to internal standard peak intensity ratio was observed for three representative fatty acids analyzed across a concentration range from 4.40 to 150 microM, with correlation coefficients between 0.986 and 0.987. The application of this method was demonstrated with the analysis of free fatty acids in nonfasted and fasted rat plasmas. A total of eight free fatty acids (14:0, 16:0, 16:1, 17:0, 18:0, 18:1, 18:2, and 20:4) were detected. The relative peak height ratios of the fatty acids to the internal standard allow quantitative measurements of the free fatty acids. It was shown that the levels of free fatty acids were higher in fasted rats than in rats in a nonfasted state. This method is simple, sensitive, and fast. Thus, it provides an appealing tool for the analysis of free fatty acids or other low-molecular weight compounds during drug discovery and/or development.
Assuntos
Ácidos Graxos não Esterificados/sangue , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Acetatos , Animais , Análise Química do Sangue/métodos , Análise Química do Sangue/normas , Ácidos Graxos não Esterificados/química , Ácidos Graxos não Esterificados/normas , Compostos Férricos , Indicadores e Reagentes , Masculino , Metaloporfirinas , Ratos , Ratos Sprague-Dawley , Padrões de Referência , Acetato de Sódio , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/normasRESUMO
The effect of the calcium ion (Ca2+) on the growth and differentiation of the mouse epidermal keratinocytes cultured in serum-free medium was investigated. It was found that the optimal level of calcium ion in the medium was about 0.2 mmol/L. Under such a culture condition the colony forming efficiency, attachment percentage, percentage of the cells with cornified envelops, and percentage of the senesced cells were measured to be about 10.8%, 30.8%, 5.1%, and 26.8%, respectively. However, the Ca2+ concentrations in the medium above 0.6 mmol/L resulted in significant differentiation and senescence of the keratinocytes, which was found to be harmful for keratinocyte growth and expansion in vitro.