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1.
Int J Mol Sci ; 24(23)2023 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-38069027

RESUMO

Rapid and accurate serological analysis of SARS-CoV-2 antibodies is important for assessing immune protection from vaccination or infection of individuals and for projecting virus spread within a population. The quartz crystal microbalance (QCM) is a label-free flow-based sensor platform that offers an opportunity to detect the binding of a fluid-phase ligand to an immobilized target molecule in real time. A QCM-based assay was developed for the detection of SARS-CoV-2 antibody binding and evaluated for assay reproducibility. The assay was cross-compared to the Roche electrochemiluminescence assay (ECLIA) Elecsys® Anti-SARS-CoV-2 serology test kit and YHLO's chemiluminescence immunoassay (CLIA). The day-to-day reproducibility of the assay had a correlation of r2 = 0.99, p < 0.001. The assay linearity was r2 = 0.96, p < 0.001, for dilution in both serum and buffer. In the cross-comparison analysis of 119 human serum samples, 59 were positive in the Roche, 52 in the YHLO, and 48 in the QCM immunoassay. Despite differences in the detection method and antigen used for antibody capture, there was good coherence between the assays, 80-100% for positive and 96-100% for negative test results. In summation, the QCM-based SARS-CoV-2 IgG immunoassay showed high reproducibility and linearity, along with good coherence with the ELISA-based assays. Still, factors including antibody titer and antigen-binding affinity may differentially affect the various assays' responses.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/diagnóstico , Técnicas de Microbalança de Cristal de Quartzo , Reprodutibilidade dos Testes , Imunoensaio/métodos , Anticorpos Antivirais , Sensibilidade e Especificidade
2.
Front Nutr ; 10: 1236153, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37781111

RESUMO

Background: A multifunctional diet (MFD) combining foods and ingredients with proven functional properties, such as fatty fish and fiber-rich foods, among others, was developed and shown to markedly reduce cardiometabolic risk-associated factors. Objective: Here, we aim at examining metabolic physiological changes associated with these improvements. Methods: Adult overweight individuals without other risk factors were enrolled in an 8-week randomized controlled intervention following a parallel design, with one group (n = 23) following MFD and one group (n = 24) adhering to a control diet (CD) that followed the caloric formula (E%) advised by the Nordic Nutritional Recommendations. Plasma metabolites and lipids were profiled by gas chromatography and ultrahigh performance liquid chromatography/mass spectrometry. Results: Weight loss was similar between groups. The MFD and CD resulted in altered levels of 137 and 78 metabolites, respectively. Out of these, 83 were uniquely altered by the MFD and only 24 by the CD. The MFD-elicited alterations in lipid levels depended on carbon number and degree of unsaturation. Conclusion: An MFD elicits weight loss-independent systematic lipid remodeling, promoting increased circulating levels of long and highly unsaturated lipids. Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT02148653?term=NCT02148653&draw=2&rank=1, NCT02148653.

3.
Obesity (Silver Spring) ; 31(10): 2530-2542, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37587639

RESUMO

OBJECTIVE: Some patients regain weight to a variable extent from 1 year after Roux-en-Y gastric bypass surgery (RYGB), though rarely reaching preoperative values. The aim of the present study was to investigate whether, when, and to what extent metabolic remission occurs. METHODS: Fasting metabolite and lipid profiles were determined in blood plasma collected from a nonrandomized intervention study involving 148 patients before RYGB and at 2, 12, and 60 months post RYGB. Both short-term and long-term alterations in metabolism were assessed. Anthropometric and clinical variables were assessed at all study visits. RESULTS: This study found that the vast majority of changes in metabolite levels occurred during the first 2 months post RYGB. Notably, thereafter the metabolome started to return toward the presurgical state. Consequently, a close-to-presurgical metabolome was observed at the time when patients reached their lowest weight and glucose level. Lipids with longer acyl chains and a higher degree of unsaturation were altered more dramatically compared with shorter and more saturated lipids, suggesting a systematic and reversible lipid remodeling. CONCLUSIONS: Remission of the metabolic state was observed prior to notable weight regain. Further and more long-term studies are required to assess whether the extent of metabolic remission predicts future weight regain and glycemic deterioration.


Assuntos
Derivação Gástrica , Humanos , Metaboloma , Antropometria , Aumento de Peso , Lipídeos
4.
Sci Rep ; 10(1): 11561, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665614

RESUMO

Identification of biomarkers associated with protection from developing diabetic complications is a prerequisite for an effective prevention and treatment. The aim of the present study was to identify clinical and plasma metabolite markers associated with freedom from vascular complications in people with very long duration of type 1 diabetes (T1D). Individuals with T1D, who despite having longer than 30 years of diabetes duration never developed major macro- or microvascular complications (non-progressors; NP) were compared with those who developed vascular complications within 25 years from diabetes onset (rapid progressors; RP) in the Scandinavian PROLONG (n = 385) and DIALONG (n = 71) cohorts. The DIALONG study also included 75 healthy controls. Plasma metabolites were measured using gas and/or liquid chromatography coupled to mass spectrometry. Lower hepatic fatty liver indices were significant common feature characterized NPs in both studies. Higher insulin sensitivity and residual ß-cell function (C-peptide) were also associated with NPs in PROLONG. Protection from diabetic complications was associated with lower levels of the glycolytic metabolite pyruvate and APOCIII in PROLONG, and with lower levels of thiamine monophosphate and erythritol, a cofactor and intermediate product in the pentose phosphate pathway as well as higher phenylalanine, glycine and serine in DIALONG. Furthermore, T1D individuals showed elevated levels of picolinic acid as compared to the healthy individuals. The present findings suggest a potential beneficial shunting of glycolytic substrates towards the pentose phosphate and one carbon metabolism pathways to promote nucleotide biosynthesis in the liver. These processes might be linked to higher insulin sensitivity and lower liver fat content, and might represent a mechanism for protection from vascular complications in individuals with long-term T1D.


Assuntos
Peptídeo C/sangue , Complicações do Diabetes/genética , Diabetes Mellitus Tipo 1/genética , Nucleotídeos/sangue , Idoso , Biomarcadores/sangue , Glicemia , Complicações do Diabetes/sangue , Complicações do Diabetes/patologia , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/patologia , Feminino , Predisposição Genética para Doença , Humanos , Resistência à Insulina/genética , Fígado/metabolismo , Masculino , Metabolômica , Pessoa de Meia-Idade , Nucleotídeos/biossíntese
5.
Diabetes ; 69(9): 2027-2035, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32527768

RESUMO

Bariatric surgery is an efficient method to induce weight loss and also, frequently, remission of type 2 diabetes (T2D). Unpaired studies have shown bariatric surgery and dietary interventions to differentially affect multiple hormonal and metabolic parameters, suggesting that bariatric surgery causes T2D remission at least partially via unique mechanisms. In the current study, plasma metabolite profiling was conducted in patients with (n = 10) and without T2D (n = 9) subjected to Roux-en-Y gastric bypass surgery (RYGB). Mixed-meal tests were conducted at baseline, after the presurgical very-low-calorie diet (VLCD) intervention, immediately after RYGB, and after a 6-week recovery period. Thereby, we could compare fasted and postprandial metabolic consequences of RYGB and VLCD in the same patients. VLCD yielded a pronounced increase in fasting acylcarnitine levels, whereas RYGB, both immediately and after a recovery period, resulted in a smaller but opposite effect. Furthermore, we observed profound changes in lipid metabolism following VLCD but not in response to RYGB. Most changes previously associated with RYGB were found to be consequences of the presurgical dietary intervention. Overall, our results question previous findings of unique metabolic effects of RYGB and suggest that the effect of RYGB on the metabolite profile is mainly attributed to caloric restriction.


Assuntos
Restrição Calórica/métodos , Diabetes Mellitus Tipo 2/cirurgia , Jejum/sangue , Derivação Gástrica/métodos , Obesidade Mórbida/cirurgia , Adulto , Glicemia/metabolismo , Carnitina/análogos & derivados , Carnitina/sangue , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Humanos , Insulina/sangue , Resistência à Insulina , Pessoa de Meia-Idade , Obesidade Mórbida/complicações , Obesidade Mórbida/metabolismo , Período Pós-Prandial
6.
Acta Diabetol ; 57(5): 597-603, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31863321

RESUMO

AIMS: The population of immigrants from the Middle East in Sweden show a higher prevalence of type 2 diabetes (T2D) compared to native Swedes. The exact reason for this is unknown. Here, we have performed metabolite profiling to investigate these differences. METHODS: Metabolite profiling was conducted in Iraqi immigrants (n = 93) and native Swedes (n = 77) using two complementary mass spectrometry-based platforms. Differences in metabolite levels were compared after adjustment for confounding anthropometric, diet and clinical variables. RESULTS: The Iraqi immigrant population were more obese (44.1 vs 24.7%, p < 0.05), but had a lower prevalence of hypertension (32.3 vs 54.8%, p < 0.01) than the native Swedish population. We detected 140 metabolites, 26 of which showed different levels between populations (q < 0.05,) after adjustment for age, sex, BMI, T2D and use of metformin. Twenty-two metabolites remained significant after further adjustment for HOMA-IR, HOMA-beta or insulin sensitivity index. Levels of polyunsaturated acylcarnitines (14:2 and 18:2) and fatty acid (18:2) were higher, whereas those of saturated and monounsaturated acylcarnitines (14:0, 18:1, and 8:1), fatty acids (12:0, 14:0, 16:0, and 18:1), uremic solutes (urate and quinate) and ketone bodies (beta-hydroxybutyrate) were lower in Iraqi immigrants. Further, levels of phospholipids were generally lower in the Iraqi immigrant population. CONCLUSIONS: Our result suggests an overall beneficial lipid profile in Iraqi immigrants, despite a higher risk to develop T2D. Higher levels of polyunsaturated fatty acids may suggest differences in dietary pattern, which in turn may reduce the risk of hypertension.


Assuntos
Diabetes Mellitus Tipo 2/sangue , Ácidos Graxos Insaturados/sangue , Hipertensão/sangue , Metabolismo dos Lipídeos , Obesidade/sangue , Estresse Oxidativo , Biomarcadores/sangue , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/etnologia , Diabetes Mellitus Tipo 2/metabolismo , Suscetibilidade a Doenças , Emigrantes e Imigrantes/estatística & dados numéricos , Feminino , Humanos , Hipertensão/epidemiologia , Hipertensão/etnologia , Hipertensão/metabolismo , Resistência à Insulina , Iraque/etnologia , Masculino , Pessoa de Meia-Idade , Obesidade/epidemiologia , Obesidade/etnologia , Obesidade/metabolismo , Prevalência , Suécia/epidemiologia , Suécia/etnologia
8.
Metabolites ; 8(4)2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30445727

RESUMO

The plasma metabolome is associated with multiple phenotypes and diseases. However, a systematic study investigating clinical determinants that control the metabolome has not yet been conducted. In the present study, therefore, we aimed to identify the major determinants of the plasma metabolite profile. We used ultra-high performance liquid chromatography (UHPLC) coupled to quadrupole time of flight mass spectrometry (QTOF-MS) to determine 106 metabolites in plasma samples from 2503 subjects in a cross-sectional study. We investigated the correlation structure of the metabolite profiles and generated uncorrelated metabolite factors using principal component analysis (PCA) and varimax rotation. Finally, we investigated associations between these factors and 34 clinical covariates. Our results suggest that liver function, followed by kidney function and insulin resistance show the strongest associations with the plasma metabolite profile. The association of specific phenotypes with several components may suggest multiple independent metabolic mechanisms, which is further supported by the composition of the associated factors.

9.
Mass Spectrom Rev ; 37(2): 229-241, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-27403762

RESUMO

Mass spectrometric epitope mapping has become a versatile method to precisely determine a soluble antigen's partial structure that directly interacts with an antibody in solution. Typical lengths of investigated antigens have increased up to several 100 amino acids while experimentally determined epitope peptides have decreased in length to on average 10-15 amino acids. Since the early 1990s more and more sophisticated methods have been developed and have forwarded a bouquet of suitable approaches for epitope mapping with immobilized, temporarily immobilized, and free-floating antibodies. While up to now monoclonal antibodies have been mostly used in epitope mapping experiments, the applicability of polyclonal antibodies has been proven. The antibody's resistance towards enzymatic proteolysis has been of key importance for the two mostly applied methods: epitope excision and epitope extraction. Sample consumption has dropped to low pmol amounts on both, the antigen and the antibody. While adequate in-solution sample handling has been most important for successful epitope mapping, mass spectrometric analysis has been found the most suitable read-out method from early on. The rapidity by which mass spectrometric epitope mapping nowadays is executed outperforms all alternative methods. Thus, it can be asserted that mass spectrometric epitope mapping has reached a state of maturity, which allows it to be used in any mass spectrometry laboratory. After 25 years of constant and steady improvements, its application to clinical samples, for example, for patient characterization and stratification, is anticipated in the near future. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:229-241, 2018.


Assuntos
Mapeamento de Epitopos/métodos , Espectrometria de Massas/métodos , Animais , Anticorpos Imobilizados/química , Epitopos/isolamento & purificação , Humanos
10.
Diabetes ; 67(3): 372-384, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29229616

RESUMO

Glucagon-like peptide 1 (GLP-1), secreted from intestinal L cells, glucose dependently stimulates insulin secretion from ß-cells. This glucose dependence prevents hypoglycemia, rendering GLP-1 analogs a useful and safe treatment modality in type 2 diabetes. Although the amino acid glutamine is a potent elicitor of GLP-1 secretion, the responsible mechanism remains unclear. We investigated how GLP-1 secretion is metabolically coupled in L cells (GLUTag) and in vivo in mice using the insulin-secreting cell line INS-1 832/13 as reference. A membrane-permeable glutamate analog (dimethylglutamate [DMG]), acting downstream of electrogenic transporters, elicited similar alterations in metabolism as glutamine in both cell lines. Both DMG and glutamine alone elicited GLP-1 secretion in GLUTag cells and in vivo, whereas activation of glutamate dehydrogenase (GDH) was required to stimulate insulin secretion from INS-1 832/13 cells. Pharmacological inhibition in vivo of GDH blocked secretion of GLP-1 in response to DMG. In conclusion, our results suggest that nonelectrogenic nutrient uptake and metabolism play an important role in L cell stimulus-secretion coupling. Metabolism of glutamine and related analogs by GDH in the L cell may explain why GLP-1 secretion, but not that of insulin, is activated by these secretagogues in vivo.


Assuntos
Células Enteroendócrinas/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Glutamato Desidrogenase/metabolismo , Glutamina/metabolismo , Mitocôndrias/enzimologia , Modelos Biológicos , Administração Retal , Animais , Catequina/administração & dosagem , Catequina/análogos & derivados , Catequina/metabolismo , Linhagem Celular , Células Enteroendócrinas/efeitos dos fármacos , Células Enteroendócrinas/enzimologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Feminino , Peptídeo 1 Semelhante ao Glucagon/sangue , Glucose/metabolismo , Glutamato Desidrogenase/antagonistas & inibidores , Glutamato Desidrogenase/química , Glutamatos/farmacologia , Insulina/sangue , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fragmentos de Peptídeos/sangue , Ratos , Análise de Célula Única
11.
Thyroid ; 27(9): 1109-1117, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28699427

RESUMO

BACKGROUND: Hyperthyroidism is associated with alterations in metabolism that are currently only partially understood. The objective of the study was to investigate changes in metabolism associated with reinstatement of euthyroidism in Swedish patients. METHODS: Eighty metabolites in plasma were profiled from 10 subjects with Graves' disease (GD) at baseline and after 9 and 15 months of treatment to reinstate euthyroidism. Thyroid parameters, thyrotropin (TSH), TSH receptor antibodies, free triiodothyronine, and free thyroxine were followed. Main findings were validated in plasma from 20 subjects with GD at baseline and at three, six, and nine months. The study was conducted at the endocrinology clinic in Malmö, Sweden. RESULTS: Euthyroidism was reinstated at three months, and thyroid status did not change further during the 15-month follow-up. This was paralleled by altered levels of 9/19 detected acylcarnitines (p < 0.05 after adjustment for multiple testing). Levels of short-chain acylcarnitines were decreased, intermediate-chain acylcarnitines elevated, and long-chain acylcarnitines unaltered. CONCLUSIONS: GD and treatment of the disease is associated with pronounced acyl chain length-dependent alterations in acylcarnitine levels. These changes may be impacted by ethnicity and or dietary differences.


Assuntos
Antitireóideos/uso terapêutico , Carnitina/análogos & derivados , Ácidos Graxos Voláteis/sangue , Doença de Graves/terapia , Terapia de Reposição Hormonal , Glândula Tireoide/efeitos dos fármacos , Tiroxina/uso terapêutico , Adulto , Carnitina/sangue , Terapia Combinada , Ácidos Graxos/sangue , Ácidos Graxos/química , Feminino , Seguimentos , Doença de Graves/sangue , Doença de Graves/etnologia , Doença de Graves/fisiopatologia , Humanos , Masculino , Metabolômica/métodos , Metimazol/uso terapêutico , Pessoa de Meia-Idade , Peso Molecular , Análise de Componente Principal , Suécia , Glândula Tireoide/fisiopatologia
12.
Diabetes ; 66(4): 806-814, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27913577

RESUMO

Latent autoimmune diabetes in adults (LADA) usually refers to GAD65 autoantibodies (GADAb)-positive diabetes with onset after 35 years of age and no insulin treatment within the first 6 months after diagnosis. However, it is not always easy to distinguish LADA from type 1 or type 2 diabetes. In this study, we examined whether metabolite profiling could help to distinguish LADA (n = 50) from type 1 diabetes (n = 50) and type 2 diabetes (n = 50). Of 123 identified metabolites, 99 differed between the diabetes types. However, no unique metabolite profile could be identified for any of the types. Instead, the metabolome varied along a C-peptide-driven continuum from type 1 diabetes via LADA to type 2 diabetes. LADA was more similar to type 2 diabetes than to type 1 diabetes. In a principal component analysis, LADA patients overlapping with type 1 diabetes progressed faster to insulin therapy than those overlapping with type 2 diabetes. In conclusion, we could not find any unique metabolite profile distinguishing LADA from type 1 and type 2 diabetes. Rather, LADA was metabolically an intermediate of type 1 and type 2 diabetes, with those patients closer to the former showing a faster progression to insulin therapy than those closer to the latter.


Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Autoimune Latente em Adultos/metabolismo , Metaboloma , Adulto , Idade de Início , Autoanticorpos/imunologia , Glicemia/metabolismo , Peptídeo C/metabolismo , Estudos de Casos e Controles , Progressão da Doença , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Glutamato Descarboxilase/imunologia , Hemoglobinas Glicadas/metabolismo , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Diabetes Autoimune Latente em Adultos/tratamento farmacológico , Diabetes Autoimune Latente em Adultos/imunologia , Masculino , Pessoa de Meia-Idade , Análise de Componente Principal , Suécia , Adulto Jovem
13.
FEBS Lett ; 590(23): 4242-4251, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27943300

RESUMO

Glycogen metabolism in ß-cells may affect downstream metabolic pathways controlling insulin release. We examined glycogen metabolism in human islets and in the rodent-derived INS-1 832/13 ß-cells and found them to express the same isoforms of key enzymes required for glycogen metabolism. Our findings indicate that glycogenesis is insulin-independent but influenced by extracellular glucose concentrations. Levels of glycogen synthase decrease with increasing glucose concentrations, paralleling accumulation of glycogen. We did not find cAMP-elicited glycogenolysis and insulin secretion to be causally related. In conclusion, our results reveal regulated glycogen metabolism in human islets and insulin-secreting cells. Whether glycogen metabolism affects insulin secretion under physiological conditions remains to be determined.


Assuntos
Glucose/farmacologia , Glicogênio/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Linhagem Celular , Colforsina/farmacologia , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Glicogênio Sintase/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina
14.
Anal Chem ; 88(1): 890-7, 2016 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-26606592

RESUMO

To obtain insight into pH change-driven molecular dynamics, we studied the higher order structure changes of protein G'e at the molecular and amino acid residue levels in solution by using nanoESI- and IM-mass spectrometry, CD spectroscopy, and protein chemical modification reactions (protein footprinting). We found a dramatic change of the overall tertiary structure of protein G'e when the pH was changed from neutral to acidic, whereas its secondary structure features remained nearly invariable. Limited proteolysis and surface-topology mapping of protein G'e by fast photochemical oxidation of proteins (FPOP) under neutral and acidic conditions reveal areas where higher order conformational changes occur on the amino-acid residue level. Under neutral solution conditions, lower oxidation occurs for residues of the first linker region, whereas greater oxidative modifications occur for amino-acid residues of the IgG-binding domains I and II. We propose a dynamic model of pH-induced structural changes in which protein G'e at neutral pH adopts an overall tight conformation with all four domains packed in a firm assembly, whereas at acidic pH, the three IgG-binding domains form an elongated alignment, and the N-terminal, His-tag-carrying domain unfolds. At the same time the individual IgG-binding domains themselves seem to adopt a more compacted fold. As the secondary structure features are nearly unchanged at either pH, interchange between both conformations is highly reversible, explaining the high reconditioning power of protein G'e-based affinity chromatography columns.


Assuntos
Proteínas de Bactérias/química , Espectrometria de Massas , Simulação de Dinâmica Molecular , Concentração de Íons de Hidrogênio , Conformação Proteica , Tripsina/metabolismo
15.
J Am Soc Mass Spectrom ; 26(3): 482-92, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25560987

RESUMO

Mass spectrometric de-novo sequencing was applied to review the amino acid sequence of a commercially available recombinant protein G´ with great scientific and economic importance. Substantial deviations to the published amino acid sequence (Uniprot Q54181) were found by the presence of 46 additional amino acids at the N-terminus, including a so-called "His-tag" as well as an N-terminal partial α-N-gluconoylation and α-N-phosphogluconoylation, respectively. The unexpected amino acid sequence of the commercial protein G' comprised 241 amino acids and resulted in a molecular mass of 25,998.9 ± 0.2 Da for the unmodified protein. Due to the higher mass that is caused by its extended amino acid sequence compared with the original protein G' (185 amino acids), we named this protein "protein G'e." By means of mass spectrometric peptide mapping, the suggested amino acid sequence, as well as the N-terminal partial α-N-gluconoylations, was confirmed with 100% sequence coverage. After the protein G'e sequence was determined, we were able to determine the expression vector pET-28b from Novagen with the Xho I restriction enzyme cleavage site as the best option that was used for cloning and expressing the recombinant protein G'e in E. coli. A dissociation constant (K(d)) value of 9.4 nM for protein G'e was determined thermophoretically, showing that the N-terminal flanking sequence extension did not cause significant changes in the binding affinity to immunoglobulins.


Assuntos
Proteínas de Bactérias/química , Mapeamento de Peptídeos/métodos , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Dados de Sequência Molecular , Peso Molecular
16.
J Mol Recognit ; 27(9): 566-74, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25042711

RESUMO

The development and application of a miniaturized affinity system for the preparation and release of intact immune complexes are demonstrated. Antibodies were reversibly affinity-adsorbed on pipette tips containing protein G´ and protein A, respectively. Antigen proteins were digested with proteases and peptide mixtures were exposed to attached antibodies; forming antibody-epitope complexes, that is, immune complexes. Elution with millimolar indole propionic acid (IPA)-containing buffers under neutral pH conditions allowed to effectively isolate the intact immune complexes in purified form. Size exclusion chromatography was performed to determine the integrity of the antibody-epitope complexes. Mass spectrometric analysis identified the epitope peptides in the respective SEC fractions. His-tag-containing recombinant human glucose-6-phosphate isomerase in combination with an anti-His-tag monoclonal antibody was instrumental to develop the method. Application was extended to the isolation of the intact antibody-epitope complex of a recombinant human tripartite motif 21 (rhTRIM21) auto-antigen in combination with a rabbit polyclonal anti-TRIM21 antibody. Peptide chip analysis showed that antibody-epitope binding of rhTRIM21 peptide antibody complexes was not affected by the presence of IPA in the elution buffer. By contrast, protein G´ showed an ion charge structure by electrospray mass spectrometry that resembled a denatured conformation when exposed to IPA-containing buffers. The advantages of this novel isolation strategy are low sample consumption and short experimental duration in addition to the direct and robust methodology that provides easy access to intact antibody-antigen complexes under neutral pH and low salt conditions for subsequent investigations.


Assuntos
Complexo Antígeno-Anticorpo/isolamento & purificação , Técnicas Imunológicas/métodos , Peptídeos/química , Complexo Antígeno-Anticorpo/imunologia , Cromatografia de Afinidade , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Epitopos/imunologia , Humanos , Proteínas Imobilizadas/metabolismo , Nanopartículas/química , Peptídeos/imunologia , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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