Protein Analysis Using a Combination of an Online Monolithic Trypsin Immobilized Enzyme Reactor and Collisionally-Activated Dissociation/Electron Transfer Dissociation Dual Tandem Mass Spectrometry

Hwang, Hyo Jin; Cho, Kun; Kim, Jin Young; Kim, Young Hwan; Oh, Han Bin

doi:10.5012/bkcs.2012.33.10.3233

Cited by 6 publications

(4 citation statements)

References 71 publications

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“…Although most of the commercially produced mass spectrometers have only one fragmentation capability CAD, different combinations of CAD (performed either in trap or RF-only quadrupole collision cell) with ECD and ETD, respectively [ 114 ] are available in new state-of-the-art instruments. This also provides higher quantification accuracy, access to complementary ion information and improved proteome coverage [ 237 ].…”

Section: Part 1 Probing the Structure Of Glycated Proteins By Masmentioning

confidence: 99%

Maillard Proteomics: Opening New Pages

Soboleva

Schmidt

Vikhnina

et al. 2017

IJMS

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Protein glycation is a ubiquitous non-enzymatic post-translational modification, formed by reaction of protein amino and guanidino groups with carbonyl compounds, presumably reducing sugars and α-dicarbonyls. Resulting advanced glycation end products (AGEs) represent a highly heterogeneous group of compounds, deleterious in mammals due to their pro-inflammatory effect, and impact in pathogenesis of diabetes mellitus, Alzheimer’s disease and ageing. The body of information on the mechanisms and pathways of AGE formation, acquired during the last decades, clearly indicates a certain site-specificity of glycation. It makes characterization of individual glycation sites a critical pre-requisite for understanding in vivo mechanisms of AGE formation and developing adequate nutritional and therapeutic approaches to reduce it in humans. In this context, proteomics is the methodology of choice to address site-specific molecular changes related to protein glycation. Therefore, here we summarize the methods of Maillard proteomics, specifically focusing on the techniques providing comprehensive structural and quantitative characterization of glycated proteome. Further, we address the novel break-through areas, recently established in the field of Maillard research, i.e., in vitro models based on synthetic peptides, site-based diagnostics of metabolism-related diseases (e.g., diabetes mellitus), proteomics of anti-glycative defense, and dynamics of plant glycated proteome during ageing and response to environmental stress.

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Section: Part 1 Probing the Structure Of Glycated Proteins By Masmentioning

confidence: 99%

Maillard Proteomics: Opening New Pages

Soboleva

Schmidt

Vikhnina

et al. 2017

IJMS

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“…Off-line configurations include trypsin membrane cartridges, magnetic or glass beads, agarose gel beads, spin-columns, etc. while on-line approaches tend to immobilize trypsin in either porous monolithic materials [20, 22, 23] or particle supports packed into LC columns or capillary electrophoresis (CE) setups [5, 24–26]. One promising approach to increasing the proteomic workflow is to create a flow-through enzyme reactor, where the proteins pass through the reactor and are digested into peptides immediately on-column.…”

Section: Introductionmentioning

confidence: 99%

Characterization of an immobilized enzyme reactor for on-line protein digestion

Moore

Hess

Jorgenson

2016

Journal of Chromatography A

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Despite the developments for faster liquid chromatographic and mass spectral detection techniques, the standard in-solution protein digestion for proteomic analyses has remained relatively unchanged. The typical in-solution trypsin protein digestion is usually the slowest part of the workflow, albeit one of the most important. The development of a highly efficient immobilized enzyme reactor (IMER) with rapid performance for on-line protein digestion would greatly decrease the analysis time involved in a proteomic workflow. Presented here is the development of a silica based IMER for on-line protein digestion, which produced rapid digestions in the presence of organic mobile phase for both model proteins and a complex sample consisting of the insoluble portion of a yeast cell lysate. Protein sequence coverage and identifications evaluated between the IMER and in-solution digestions were comparable. Overall, for a yeast cell lysate with only a 10 sec volumetric residence time on-column, the IMER identified 507 proteins while the in-solution digestion identified 490. There were no significant differences observed based on identified protein’s molecular weight or isoelectric point between the two digestion methods. Implementation of the IMER into the proteomic workflow provided similar protein identification results, automation for sample analysis, and reduced the analysis time by 15 hr.

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“…IMERs come in different forms: packed, monolithic, and open-tubular. Each with its unique advantages and challenges, relating to fabrication time, equipment requirements, and enzyme surface area [19][20][21][22][23][24][25][26][27][28]. Open-tubular IMERs (OT-IMER) stand out for their useability, speed, and economic efficiency of fabrication, along with their potential for automation and enhanced reproducibility while minimizing operator handling [15,29].…”

Section: Introductionmentioning

confidence: 99%

Capillary Electrophoresis-Based Immobilized Enzyme Microreactor Utilizing Pepsin for Bottom-Up Proteomics

Ghafourifar,

Frey,

Arad

et al. 2024

Preprint

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In this study, we introduced a novel method for pepsin immobilization through investigation into three different coupling reactions, aiming to simplify peptide mapping. We initially determined the optimal enzyme-to-substrate ratio to be 1:0.1 (mass:mass) for highest peptidic peak count via UV-detection. The study began with three coupling strategies, the triethoxysilylbutyraldehyde derivative, 4-triethoxysilylbutanoic acid (4-TESBA) approach proving most effective. The resultant coupled enzyme particles (CEPs) showcased robustness and fast digestion at varying temperatures. We streamlined the CEP wash procedure from our previous work, while maintaining the quality of digestions. The physical size of CEPs did not correlate to digestion efficiency, providing insights for potential cost-saving in enzyme utilization. Further optimization led to an immobilization efficiency of 50.8 ± 7.7% (SEM) as validated by Bradford’s assay. Adapting this method for in-situ immobilized enzyme microreactor (IMER) fabrication, we discovered that 4-TESBA could dual-serve by functionalizing the silica capillary’s inner wall while simultaneously acting as an enzyme coupler, plus being a safer alternative to 3-(Aminopropyl)triethoxysilane (APTES). A dipeptide was successfully fully cleaved, and a variety of proteins were digested with the IMER. The bovine serum albumin (BSA) digestion through the IMER, mirroring CEP digestion conditions, yielded a 33-40% primary sequence coverage per LC-MS/MS analysis in as short as 15 minutes. Our findings underscore the potential of our method in both CEP and IMER fabrication scenarios, paving the way for enhanced analysis and a reduction in enzyme usage, thereby contributing to more cost-effective and timely proteomic investigations.

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Protein Analysis Using a Combination of an Online Monolithic Trypsin Immobilized Enzyme Reactor and Collisionally-Activated Dissociation/Electron Transfer Dissociation Dual Tandem Mass Spectrometry

Cited by 6 publications

References 71 publications

Maillard Proteomics: Opening New Pages

Maillard Proteomics: Opening New Pages

Characterization of an immobilized enzyme reactor for on-line protein digestion

Capillary Electrophoresis-Based Immobilized Enzyme Microreactor Utilizing Pepsin for Bottom-Up Proteomics

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