Liquid-chromatographic and mass-spectrometric identification of lens proteins using microwave-assisted digestion with trypsin-immobilized magnetic nanoparticles
“…The largest number of experiments was carried out for the sample (B) because it contained the biological material obtained from 11 lenses. The amount of reagents for reduction of the disulfide bridges and hydrolysis was calculated in relation to the proteins content in one lens according to the literature [ 28 ]. The preparation of the biological material was optimized to obtain the largest number of the modified peptides.…”
Section: Resultsmentioning
confidence: 99%
“…The experiments consisted of the same steps which were performed in different order. The amounts of reagents (trypsin and dithiothreitol) were calculated on the basis of the amount of proteins isolated from one lens according to the literature [ 28 ]. Fig.…”
Glycation of α-crystallin is responsible for age- and diabetic-related cataracts, which are the main cause of blindness worldwide. We optimized the method of identification of lysine residues prone to glycation using the combination of LC-MS, isotopic labeling, and modified synthetic peptide standards with the glycated lysine derivative (Fmoc-Lys(i,i-Fru,Boc)-OH). The in vitro glycation of bovine lens α-crystallin was conducted by optimized method with the equimolar mixture of [12C6]- and [13C6]d-glucose. The in vivo glycation was studied on human lens crystallin. The glycated protein was subjected to proteolysis and analyzed using LC-MS. The results of in vitro and in vivo glycation of α-crystallin reveal a different distribution of the modified lysine residues. More Amadori products were detected as a result of the in vitro reaction due to forced glycation conditions. The developed method allowed us to identify the glycation sites in crystallin from eye lenses obtained from patients suffering from the cataract. We identified K166 in the A chain and K166 in the B chain of α-crystallin as major glycation sites during the in vitro reaction. We found also two in vivo glycated lysine residues: K92 in the B chain and K166 in the A chain, which are known as locations for Amadori products. These modification sites were confirmed by the LC-MS experiment using two synthetic standards. This study demonstrates the applicability of the LC-MS methods combined with the isotopic labeling and synthetic peptide standards for analysis of post-translational modifications in the biological material.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-015-8487-7) contains supplementary material, which is available to authorized users.
“…The largest number of experiments was carried out for the sample (B) because it contained the biological material obtained from 11 lenses. The amount of reagents for reduction of the disulfide bridges and hydrolysis was calculated in relation to the proteins content in one lens according to the literature [ 28 ]. The preparation of the biological material was optimized to obtain the largest number of the modified peptides.…”
Section: Resultsmentioning
confidence: 99%
“…The experiments consisted of the same steps which were performed in different order. The amounts of reagents (trypsin and dithiothreitol) were calculated on the basis of the amount of proteins isolated from one lens according to the literature [ 28 ]. Fig.…”
Glycation of α-crystallin is responsible for age- and diabetic-related cataracts, which are the main cause of blindness worldwide. We optimized the method of identification of lysine residues prone to glycation using the combination of LC-MS, isotopic labeling, and modified synthetic peptide standards with the glycated lysine derivative (Fmoc-Lys(i,i-Fru,Boc)-OH). The in vitro glycation of bovine lens α-crystallin was conducted by optimized method with the equimolar mixture of [12C6]- and [13C6]d-glucose. The in vivo glycation was studied on human lens crystallin. The glycated protein was subjected to proteolysis and analyzed using LC-MS. The results of in vitro and in vivo glycation of α-crystallin reveal a different distribution of the modified lysine residues. More Amadori products were detected as a result of the in vitro reaction due to forced glycation conditions. The developed method allowed us to identify the glycation sites in crystallin from eye lenses obtained from patients suffering from the cataract. We identified K166 in the A chain and K166 in the B chain of α-crystallin as major glycation sites during the in vitro reaction. We found also two in vivo glycated lysine residues: K92 in the B chain and K166 in the A chain, which are known as locations for Amadori products. These modification sites were confirmed by the LC-MS experiment using two synthetic standards. This study demonstrates the applicability of the LC-MS methods combined with the isotopic labeling and synthetic peptide standards for analysis of post-translational modifications in the biological material.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-015-8487-7) contains supplementary material, which is available to authorized users.
“…The enzyme was removed and reused by using an external magnetic field. A similar approach has been described to study the proteins of human lens tissue [20]. The method allowed the identification of 26 proteins compared with 11 proteins identified with traditional insolution digestion method.…”
Section: High Throughput Biomolecular Separation Assays Using Nanoparmentioning
confidence: 98%
“…Trypsin is widely used for protein digestion as it yields tryptic peptides of an appropriate size for mass spectrometry (MS) analysis, but the process requires several hours. The use of magnetic NPs with immobilized trypsin has been proposed for the reduction of the digestion time from hours to minutes or seconds [19][20][21]. Thus, a method involving microwave-assisted protein digestion has been described in which magnetic NPs are used as support for enzyme immobilization and, also, as absorbents of the microwave radiation, improving the efficiency of the process [19].…”
Section: High Throughput Biomolecular Separation Assays Using Nanoparmentioning
An overview of the usefulness of different nanoparticles to improve the features of high throughput separation and individual and multiplexed detection bioassays is presented. Although the development of microarray and microfluidic systems has expanded the capabilities of these high throughput assays, the combined use of NPs and these devices has provided them with new applications in drug discovery, proteomic and genomic studies, and clinical diagnosis. This article reviews the wide application field of magnetic, gold, silver, semiconductor and other nanoparticles in high throughput bioassays. Also, the versatility of the detection systems described shows that NPs are useful alternatives to fluorescent dyes, which are often used in these assays.
“…Microwave radiation effects may be purely thermal, but a certain agitation in molecular structures and motions, caused by microwave radiation, may also be involved in the enzymatic effects of this technique. Some reports have described rapid proteolysis achieved through a combination of immobilized trypsin and microwave radiation . The application of microwave‐assisted digestion with automation by a robotic system showed highly effective proteolysis and high‐throughput analysis (96 samples within 80 min) .…”
Section: Proteolysis Under Unusual Conditionsmentioning
Proteolysis is a key step in proteomic studies integrated with MS analysis but the conventional method of in-solution digestion is limited by time-consuming procedures and low sensitivity. Furthermore, obtaining reliable peptide maps and meaningful sequence data using MS analysis requires not only the separation of the digested peptides but also strictly defined proteolysis conditions. Recently, various immobilized-enzyme reactors have been developed for highly efficient proteolysis in MS-based proteomic analysis. This review focuses on the proteolysis step using protease-immobilized reactors and rapid analysis of protein sequences. We describe the preparation of enzyme reactors by several techniques and protein digestion under unusual conditions. Analysis of posttranslational modifications by enzyme reactors prepared using our immobilization method is presented as a model application. Analysis systems using immobilized-enzyme reactors are expected to become useful tools for proteomic studies and diverse applications in biotechnology.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
