Protein Glycation in Diabetes as Determined by Mass Spectrometry

Diabetes is a common endocrine disorder characterized by hyperglycemia leading to nonenzymatic glycation of proteins, responsible for chronic complications. The development of mass spectrometric techniques able to give highly specific and reliable results in proteome field is of wide interest for physicians, giving them new tools to monitor the disease progression and the possible complications related to diabetes, as well as the effectiveness of therapeutic treatments. This paper reports and discusses some of the data pertaining protein glycation in diabetic subjects obtained by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). The preliminary studies carried out byin vitroprotein glycation experiments show clear differences in molecular weight of glycated and unglycated proteins. Then, the attention was focused on plasma proteins human serum albumin (HSA) and immunoglobulin G (IgG). Enzymatic degradation products ofin vitroglycated HSA were studied in order to simulate thein vivoenzymatic digestion of glycated species by the immunological system leading to the highly reactive advanced glycation end-products (AGEs) peptides. Further studies led to the evaluation of glycated Apo A-I and glycated haemoglobin levels. A different MALDI approach was employed for the identification of markers of disease in urine samples of healthy, diabetic, nephropathic, and diabetic-nephropathic subjects.

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An Electrochemical Chip to Monitor In Vitro Glycation of Proteins and Screening of Antiglycation Potential of Drugs

Pharmaceutics ◽

10.3390/pharmaceutics12111011 ◽

2020 ◽

Vol 12 (11) ◽

pp. 1011

Author(s):

Zeeshan A. Khan ◽

Seungkyung Park

Keyword(s):

Inhibitory Concentration ◽

Strong Relationship ◽

Protein Glycation ◽

Capacitance Measurement ◽

Efficacy Evaluation ◽

Chronic Complications ◽

Glycation End Products ◽

Significant Change ◽

Selection Of

Hyperglycemia and the production of advanced glycation end products (AGEs) are the primary factors for the development of chronic complications in diabetes. The level of protein glycation is proportional to the glucose concentration and represents mean glycemia. In this study, we present an electrochemical chip-based method for in vitro glycation monitoring and the efficacy evaluation of an antiglycation compound. An electrochemical chip consisting of five microchambers and embedded microelectrodes was designed for parallel measurements of capacitance signals from multiple solutions at different concentrations. The feasibility of glycation monitoring was then investigated by measuring the capacitance signal at 0.13 MHz with bovine serum albumin and gelatin samples in the presence of various glucose concentrations over 28 days. A significant change in the capacitance due to protein glycation was observed through measurements conducted within 30 s and 21 days of incubation. Finally, we demonstrated that the chip-based capacitance measurement can be utilized for the selection of an antiglycation compound by supplementing the protein solution and hyperglycemic concentration of glucose with an inhibitory concentration of the standard antiglycation agent aspirin. The lack of a significant change in the capacitance over 28 days proved that aspirin is capable of inhibiting protein glycation. Thus, a strong relationship exists between glycation and capacitance, suggesting the application of an electrochemical chip for evaluating glycation and novel antiglycation agents.

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Localization of sunitinib in in vivo animal and in vitro experimental models by MALDI mass spectrometry imaging

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-014-8350-2 ◽

2014 ◽

Vol 407 (8) ◽

pp. 2245-2253 ◽

Cited By ~ 10

Author(s):

James J. Connell ◽

Yutaka Sugihara ◽

Szilvia Török ◽

Balázs Döme ◽

József Tóvári ◽

...

Keyword(s):

Mass Spectrometry ◽

Mass Spectrometry Imaging ◽

Maldi Mass Spectrometry ◽

Experimental Models ◽

Maldi Mass Spectrometry Imaging

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A fluorogenic assay for methylglyoxal

Biochemical Society Transactions ◽

10.1042/bst20140028 ◽

2014 ◽

Vol 42 (2) ◽

pp. 548-555 ◽

Cited By ~ 14

Author(s):

Fozia Shaheen ◽

Anatoly Shmygol ◽

Naila Rabbani ◽

Paul J. Thornalley

Keyword(s):

Degradation Products ◽

Live Cells ◽

Cell Extracts ◽

Glycation End Products ◽

Fluorogenic Probes ◽

Co Detection ◽

High Throughput Assay ◽

Dialysis Fluids

MG (methylglyoxal) is a potent glycating agent and an endogenous reactive dicarbonyl metabolite formed in all live cells and organisms. It is an important precursor of AGEs (advanced glycation end-products) and is implicated in aging and disease. MG is assayed by derivatization by 1,2-diaminobenzene derivatives in cell extracts. Such assays are not applicable to high sample throughput, subcellular, live-cell and in vivo estimations. The use of fluorogenic probes designed for NO (nitric oxide) detection in biological samples and living cells has inadvertently provided probes for the detection of dicarbonyls such as MG. We describe the application of DAF-2 (4,5-diaminofluorescein) and DAR-1 (4,5-diaminorhodamine) for the detection of MG in cell-free systems and application for high-throughput assay of glyoxalase activity and assay of glucose degradation products in peritoneal dialysis fluids. DAF-2 and DAR-1, as for related BODIPY probes, do not have sufficient sensitivity to detect MG in live cells. Care will also be required to control for NO and dehydroascorbate co-detection and interference from peroxidase catalysing the degradation of probes to MG and glyoxal. Fluorogenic detection of MG, however, has great potential to facilitate the assay of MG and to advance towards that capability of imaging this product in live cells in vitro and small animals in vivo.

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Protein glycation in vivo: functional and structural effects on yeast enolase

Biochemical Journal ◽

10.1042/bj20080632 ◽

2008 ◽

Vol 416 (3) ◽

pp. 317-326 ◽

Cited By ~ 33

Author(s):

Ricardo A. Gomes ◽

Luís M. A. Oliveira ◽

Mariana Silva ◽

Carla Ascenso ◽

Alexandre Quintas ◽

...

Keyword(s):

Advanced Glycation End Products ◽

Specific Protein ◽

Protein Glycation ◽

Advanced Glycation ◽

Structure And Stability ◽

Glycation End Products ◽

End Products ◽

Activity Loss

Protein glycation is involved in structure and stability changes that impair protein functionality, which is associated with several human diseases, such as diabetes and amyloidotic neuropathies (Alzheimer's disease, Parkinson's disease and Andrade's syndrome). To understand the relationship of protein glycation with protein dysfunction, unfolding and β-fibre formation, numerous studies have been carried out in vitro. All of these previous experiments were conducted in non-physiological or pseudo-physiological conditions that bear little to no resemblance to what may happen in a living cell. In vivo, glycation occurs in a crowded and organized environment, where proteins are exposed to a steady-state of glycation agents, namely methylglyoxal, whereas in vitro, a bolus of a suitable glycation agent is added to diluted protein samples. In the present study, yeast was shown to be an ideal model to investigate glycation in vivo since it shows different glycation phenotypes and presents specific protein glycation targets. A comparison between in vivo glycated enolase and purified enolase glycated in vitro revealed marked differences. All effects regarding structure and stability changes were enhanced when the protein was glycated in vitro. The same applies to enzyme activity loss, dimer dissociation and unfolding. However, the major difference lies in the nature and location of specific advanced glycation end-products. In vivo, glycation appears to be a specific process, where the same residues are consistently modified in the same way, whereas in vitro several residues are modified with different advanced glycation end-products.

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High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma

Molecules ◽

10.3390/molecules25184071 ◽

2020 ◽

Vol 25 (18) ◽

pp. 4071

Author(s):

Elisa Maffioli ◽

Zhenze Jiang ◽

Simona Nonnis ◽

Armando Negri ◽

Valentina Romeo ◽

...

Keyword(s):

Mass Spectrometry ◽

Proteolytic Activity ◽

High Resolution Mass Spectrometry ◽

Degradation Products ◽

Quantitative Information ◽

Peptidase Activity ◽

Vitro Assay ◽

Unbiased Manner

Proteomic technologies have identified 234 peptidases in plasma but little quantitative information about the proteolytic activity has been uncovered. In this study, the substrate profile of plasma proteases was evaluated using two nano-LC-ESI-MS/MS methods. Multiplex substrate profiling by mass spectrometry (MSP-MS) quantifies plasma protease activity in vitro using a global and unbiased library of synthetic peptide reporter substrates, and shotgun peptidomics quantifies protein degradation products that have been generated in vivo by proteases. The two approaches gave complementary results since they both highlight key peptidase activities in plasma including amino- and carboxypeptidases with different substrate specificity profiles. These assays provide a significant advantage over traditional approaches, such as fluorogenic peptide reporter substrates, because they can detect active plasma proteases in a global and unbiased manner, in comparison to detecting select proteases using specific reporter substrates. We discovered that plasma proteins are cleaved by endoproteases and these peptide products are subsequently degraded by amino- and carboxypeptidases. The exopeptidases are more active and stable in plasma and therefore were found to be the most active proteases in the in vitro assay. The protocols presented here set the groundwork for studies to evaluate changes in plasma proteolytic activity in shock.

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Comparison of High Purity Factor IX Concentrates and a Prothrombin Complex Concentrate in a Canine Model of Thrombogenicity

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646468 ◽

1991 ◽

Vol 66 (05) ◽

pp. 609-613 ◽

Cited By ~ 14

Author(s):

I R MacGregor ◽

J M Ferguson ◽

L F McLaughlin ◽

T Burnouf ◽

C V Prowse

Keyword(s):

High Purity ◽

Time Course ◽

Prothrombin Complex Concentrate ◽

Degradation Products ◽

Canine Model ◽

Factor Ix ◽

In Vitro Tests ◽

Albumin Infusion

SummaryA non-stasis canine model of thrombogenicity has been used to evaluate batches of high purity factor IX concentrates from 4 manufacturers and a conventional prothrombin complex concentrate (PCC). Platelets, activated partial thromboplastin time (APTT), fibrinogen, fibrin(ogen) degradation products and fibrinopeptide A (FPA) were monitored before and after infusion of concentrate. Changes in FPA were found to be the most sensitive and reproducible indicator of thrombogenicity after infusion of batches of the PCC at doses of between 60 and 180 IU/kg, with a dose related delayed increase in FPA occurring. Total FPA generated after 100-120 IU/kg of 3 batches of PCC over the 3 h time course was 9-12 times that generated after albumin infusion. In contrast the amounts of FPA generated after 200 IU/kg of the 4 high purity factor IX products were in all cases similar to albumin infusion. It was noted that some batches of high purity concentrates had short NAPTTs indicating that current in vitro tests for potential thrombogenicity may be misleading in predicting the effects of these concentrates in vivo.

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Accumulation of Macromolecular Particles in the Reticuloendothelial System (RES) Mediated by Platelet Aggregation and Disaggregation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651386 ◽

1969 ◽

Vol 22 (03) ◽

pp. 496-507 ◽

Cited By ~ 17

Author(s):

W.G van Aken ◽

J Vreeken

Keyword(s):

Platelet Aggregation ◽

Degradation Products ◽

Reticuloendothelial System ◽

Caproic Acid ◽

Carbon Particles ◽

Carbon Clearance ◽

Aggregation And Disaggregation ◽

Platelet Aggregates

SummaryCarbon particles cause platelet aggregation in vitro and in vivo. Prior studies established that substances which modify thrombocyte aggregation also influence the rate at which carbon is cleared from the blood.This study was performed in order to elucidate the mechanism by which the carbon-platelet aggregates specifically accumulate in the RES.Activation of fibrinolysis by urokinase or streptokinase reduced the carbon clearance rate, probably due to generated fibrinogen degradation products (FDP). Isolated FDP decreased the carbon clearance and caused disaggregation of platelets and particles in vitro. Inhibition of fibrinolysis by epsilon-amino-caproic acid (EACA), initially accelerated the disappearance of carbon and caused particle accumulation outside the RES, predominantly in the lungs. It is supposed that platelet aggregation and locally activated fibrinolysis act together in the clearance of particles. In the normal situation the RES with its well known low fibrinolytic activity, becomes the receptor of the particles.

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The Effects of Brinolase (Fibrinolytic Enzyme from Aspergillus Oryzae) on Platelet Aggregation of Dog and Man

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649039 ◽

1972 ◽

Vol 28 (01) ◽

pp. 031-048 ◽

Cited By ~ 4

Author(s):

W. H. E Roschlau ◽

R Gage

Keyword(s):

Platelet Aggregation ◽

Aspergillus Oryzae ◽

Degradation Products ◽

Blood Platelet ◽

Human Platelets ◽

Fibrinolytic Enzyme ◽

Inhibitory Effects ◽

Blood Platelet Aggregation

SummaryInhibition of blood platelet aggregation by brinolase (fibrinolytic enzyme from Aspergillus oryzae) has been demonstrated with human platelets in vitro and with dog platelets in vivo and in vitro, using both ADP and collagen as aggregating stimuli. It is suggested that the optimal inhibitory effects of brinolase occur indirectly through the generation of plasma fibrinogen degradation products, without compromising platelet viability, rather than by direct proteolysis of platelet structures.

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In vitro assessment of food-derived-glucose bioaccessibility and bioavailability in bicameral cell culture system

Turkish Journal of Biochemistry ◽

10.1515/tjb-2020-0082 ◽

2020 ◽

Vol 45 (5) ◽

pp. 631-637

Author(s):

Cansu Ozel-Tasci ◽

Gozde Pilatin ◽

Ozgur Edeer ◽

Sukru Gulec

Keyword(s):

Cell Culture ◽

Culture System ◽

Metabolic Diseases ◽

Enzymatic Digestion ◽

Cell Culture System ◽

Culture Studies ◽

Experimental Approaches ◽

In Vitro Assessment

AbstractBackgroundFunctional foods can help prevent metabolic diseases, and it is essential to evaluate functional characteristics of foods through in vitro and in vivo experimental approaches.ObjectiveWe aimed to use the bicameral cell culture system combined with the in vitro digestion to evaluate glucose bioavailability.Materials and methodsCake, almond paste, and pudding were modified by adding fiber and replacing sugar with sweeteners and polyols. Digestion process was modeled in test tubes. Rat enterocyte cells (IEC-6) were grown in a bicameral cell culture system to mimic the physiological characteristics of the human intestine. The glucose bioaccessibility and cellular glucose efflux were measured by glucose oxidase assay.Results and discussionThe glucose bioaccessibilities of modified foods were significantly lower (cake: 2.6 fold, almond paste: 9.2 fold, pudding 2.8 fold) than the controls. Cellular glucose effluxes also decreased in the modified cake, almond paste, and pudding by 2.2, 4, and 2 fold respectively compared to their controls.ConclusionOur results suggest that combining in vitro enzymatic digestion with cell culture studies can be a practical way to test in vitro glucose bioaccessibility and bioavailability in functional food development.

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Influence of Dopamine on Fluorescent Advanced Glycation End Products Formation Using Drosophila melanogaster

Biomolecules ◽

10.3390/biom11030453 ◽

2021 ◽

Vol 11 (3) ◽

pp. 453

Author(s):

Ana Filošević Vujnović ◽

Katarina Jović ◽

Emanuel Pištan ◽

Rozi Andretić Waldowski

Keyword(s):

Drosophila Melanogaster ◽

Advanced Glycation End Products ◽

Model Organism ◽

Covalent Modification ◽

Advanced Glycation ◽

Biomarkers Of Aging ◽

Glycation End Products ◽

End Products

Non-enzymatic glycation and covalent modification of proteins leads to Advanced Glycation End products (AGEs). AGEs are biomarkers of aging and neurodegenerative disease, and can be induced by impaired neuronal signaling. The objective of this study was to investigate if manipulation of dopamine (DA) in vitro using the model protein, bovine serum albumin (BSA), and in vivo using the model organism Drosophila melanogaster, influences fluorescent AGEs (fAGEs) formation as an indicator of dopamine-induced oxidation events. DA inhibited fAGEs-BSA synthesis in vitro, suggesting an anti-oxidative effect, which was not observed when flies were fed DA. Feeding flies cocaine and methamphetamine led to increased fAGEs formation. Mutants lacking the dopaminergic transporter or the D1-type showed further elevation of fAGEs accumulation, indicating that the long-term perturbation in DA function leads to higher production of fAGEs. To confirm that DA has oxidative properties in vivo, we fed flies antioxidant quercetin (QUE) together with methamphetamine. QUE significantly decreased methamphetamine-induced fAGEs formation suggesting that the perturbation of DA function in vivo leads to increased oxidation. These findings present arguments for the use of fAGEs as a biomarker of DA-associated neurodegenerative changes and for assessment of antioxidant interventions such as QUE treatment.

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