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Comparison of gel and urea methods of digestion for proteomic analysis of organ perfusion solutions

ABSTRACT: The aim of this study was to develop an optimized Shotgun proteomics method for perusate by comparing and identifying the most effective and reliable techniques for protein estimation and digestion.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Sadr Shaheed

LAB HEAD: Dr Sadr ul Shaheed

PROVIDER: PXD040856 | Pride | 2024-01-26

REPOSITORIES: Pride

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			Action	DRS
	In-gel-kidney-HMP-02-P1-FD09177341.raw	Raw
	In-gel-kidney-HMP-02-P2-FD09177356.raw	Raw
	In-gel-kidney-HMP-02-P2-FD09177913.raw	Raw
	In-gel-kidney-HMP-02-P3-FD01269744.raw	Raw
	In-gel-kidney-HMP-02-P3-FD06420623.raw	Raw

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Publications

Comparison of in-gel and in-solution proteolysis in the proteome profiling of organ perfusion solutions.

Snashall Corinna M CM Sutton Chris W CW Faro Letizia Lo LL Ceresa Carlo C Ploeg Rutger R Shaheed Sadr Ul SU

Clinical proteomics 20231115 1

<h4>Purpose</h4>The organ perfusion solution (perfusate), collected at clinically and temporally significant stages of the organ preservation and transplantation process, provides a valuable insight into the biological status of an organ over time and prior to reperfusion (transplantation) in the recipient. The objective of this study was to assess two bottom-up proteomics workflows for the extraction of tryptic peptides from the perfusate.<h4>Experimental design</h4>Two different kinds of perfu ...[more]

PMID: 37968584

Similar Datasets

Comparison of in-gel and in-solution proteolysis in the proteome profiling of organ perfusion solutions.

Project description:PurposeThe organ perfusion solution (perfusate), collected at clinically and temporally significant stages of the organ preservation and transplantation process, provides a valuable insight into the biological status of an organ over time and prior to reperfusion (transplantation) in the recipient. The objective of this study was to assess two bottom-up proteomics workflows for the extraction of tryptic peptides from the perfusate.Experimental designTwo different kinds of perfusate samples from kidney and liver trials were profiled using liquid chromatography-mass spectrometry (LC-MS/MS). The preparation of clean peptide mixtures for downstream analysis was performed considering different aspects of sample preparation; protein estimation, enrichment, in-gel and urea-based in-solution digestion.ResultsIn-solution digestion of perfusate allowed identification of the highest number of peptides and proteins with greater sequence coverage and higher confidence data in kidney and liver perfusate. Key pathways identified by gene ontology analysis included complement, coagulation and antioxidant pathways, and a number of biomarkers previously linked to ischemia-reperfusion injury were also observed in perfusate.ConclusionsThis study showed that in-solution digestion is a more efficient method for LC-MS/MS analysis of kidney and liver organ perfusion solutions. This method is also quicker and easier than in-gel digestion, allowing for greater sample throughput, with fewer opportunities for experimental error or peptide loss.

| S-EPMC10648346 | biostudies-literature

Hit-Gel: Streamlining in-gel protein digestion for high-throughput proteomics experiments.

Project description:In-gel digestion has been used as a standard method for the preparation of protein samples for mass spectrometry analysis for over 25 years. Traditional in gel-digestion procedures require extensive sample handling, are prone to contamination and not compatible with high-throughput sample preparation. To address these shortcomings, we have modified the conventional in-gel digestion procedure for high-throughput proteomics studies. The modified method, termed "High Throughput in Gel digestion" (HiT-Gel), is based on a 96-well plate format which results in a drastic reduction in labour intensity and sample handling. Direct comparison revealed that HiT-Gel reduces technical variation and significantly decreases sample contamination over the conventional in-gel digestion method. HiT-Gel also produced superior results when a single protein band was excised from a gel and processed by in-gel digestion. Moreover, we applied Hit-Gel for a mass spectrometry analysis of Arabidopsis thaliana protein complexes separated by native PAGE in 24 fractions and four biological replicates. We show that the high throughput capacity of HiT-Gel facilitates large scale studies with high sample replication or detailed fractionation. Our method can easily be implemented as it does not require specialised laboratory equipment.

| S-EPMC5988721 | biostudies-literature

Perfluorooctanoate in Aqueous Urea Solutions: Micelle Formation, Structure, and Microenvironment.

Project description:Fluorinated surfactants are used in a wide range of applications that involve aqueous solvents incorporating various additives. The presence of organic compounds such as urea is expected to affect the self-assembly of fluorinated surfactants, however, very little is known about this. We investigated the effect of urea on the micellization in water of the common fluorinated surfactant ammonium perfluorooctanoate (APFO), and on the structure and microenvironment of the micelles that APFO forms. Addition of urea to aqueous APFO solution decreased the critical micellization concentration (CMC) and increased the counterion dissociation. The observed increase in surface area per APFO headgroup and decrease in packing density at the micelle surface suggest the localization of urea at the micelle surface in a manner that reduces headgroup repulsions. Micropolarity data further support this picture. The results presented here indicate that significant differences exist between urea effects on fluorinated surfactant and on hydrocarbon surfactant micellization in aqueous solution. For example, the CMC of sodium dodecyl sulfate (SDS) increased with urea addition, while the increase in surface area per headgroup and packing density of SDS with urea addition are much lower than those observed for APFO. This study informs fluorinated surfactant fate and transport in the environment, and also applications involving aqueous media in which urea or similar additives are present.

| S-EPMC6888096 | biostudies-literature

Phenylglyoxaldehyde-Functionalized Polymeric Sorbents for Urea Removal from Aqueous Solutions.

Project description:For realization of a wearable artificial kidney based on regeneration of a small volume of dialysate, efficient urea removal from dialysate is a major challenge. Here a potentially suitable polymeric sorbent based on phenylglyoxaldehyde (PGA), able to covalently bind urea under physiological conditions, is described. Sorbent beads containing PGA groups were obtained by suspension polymerization of either styrene or vinylphenylethan-1-one (VPE), followed by modification of the aromatic groups of poly(styrene) and poly(VPE) into PGA. It was found that PGA-functionalized sorbent beads had maximum urea binding capacities of 1.4-2.2 mmol/g and removed ∼0.6 mmol urea/g in 8 h at 37 °C under static conditions from urea-enriched phosphate-buffered saline, conditions representative of dialysate regeneration. This means that the daily urea production of a dialysis patient can be removed with a few hundred grams of this sorbent which, is an important step forward in the development of a wearable artificial kidney.

| S-EPMC7027168 | biostudies-literature

Centrifugal methods and devices for rapid in-gel digestion of proteins.

Project description:Modern proteomic research frequently relies upon separation of proteins in a polyacrylamide gel matrix followed by in-gel enzymatic digestion and extraction of peptides for subsequent analysis by MS. In this work, we propose a novel semi-automated method of mechanical processing of gel bands by passing these bands through a specially designed centrifugal device termed a Gel Shredder prior to digestion and extraction of peptides. Such a device allows integrated washing, destaining and shredding of gel bands into uniform blocks of controlled size, approximately 150-300 microm, prior to the enzymatic digestion and extraction of peptides. Shredding into uniform blocks increases the surface area of the gel pieces and promotes improved gel rehydration, allowing improved diffusion of the proteolytic enzymes and solvent into the gel lattice. We demonstrate that the new method substantially reduces the time spent on tedious manual handling of gel bands, while minimizing the risk of sample contamination. The performance of the Gel Shredder has been compared with a conventional in-gel digestion protocol using several standard proteins and a complex proteomic sample in terms of relative quantitation by either MALDI-TOF/TOF or nanoLC-ESI IT-Fourier transformation ion cyclotron resonance MS. It is shown that significant time savings and improved peptide recovery can be obtained for many proteins using the Gel Shredder compared with the traditional in-gel digestion protocol.

| S-EPMC2907145 | biostudies-literature

Dynamical Effects of Trimethylamine N-Oxide on Aqueous Solutions of Urea.

Project description:Trimethylamine N-oxide (TMAO) stabilizes protein structures, whereas urea destabilizes proteins, and their opposing effects can be counteracted at a 1:2 ratio of TMAO to urea. To investigate how they affect solution dynamics, molecular dynamics simulations have been carried out for aqueous solutions of TMAO and urea at different concentrations. In the binary solutions, urea mainly slows the diffusion of waters that are hydrogen bonded to it (i.e., hydration water), whereas TMAO dramatically slows the diffusion of both hydration water and bulk water because of long-lived TMAO-water hydrogen bonds. In the ternary solutions, because TMAO decreases the diffusion rate of bulk water, the lifetimes of not only water-water but also urea-water hydrogen bonds increase. In addition, the constant forming and breaking of short lifetime hydrogen bonds between urea and water appears to impart energy into the bulk, whereas the long lifetime hydrogen bonds between TMAO and water slows down the bulk, resulting in the compensating effects on bulk water in the ternary solution. This suggests that the counteracting effects of TMAO on urea denaturation may be both to make longer lived hydrogen bonds to water and to counter the energizing effects of urea on bulk water.

| S-EPMC7085122 | biostudies-literature

Advances in Perfusion Systems for Solid Organ Preservation.

Project description:In the past, a diagnosis of organ failure would essentially be a death sentence for patients. With improved techniques for organ procurement and surgical procedures, transplantations to treat organ failure have become standard medical practice. However, while the demand for organs has skyrocketed, the donor pool has not kept pace leading to long recipient waiting lists. Organ preservation provides a means to increase the number of available transplantable organs. However, there are significant drawbacks associated with cold storage, the current gold standard. To address the short-comings due to diffusional limitations, engineers have developed cold perfusion systems. More recently, there has been a significant trend towards the development of near-normothermic systems to enhance the functional preservation of solid organs including livers, lungs, hearts, kidneys, and vascularized composite allotransplants. Here we review recent advances in the development of perfusion systems for the preservation of solid organs. We provide a brief history of organ transplantation, the limitations of existing systems, and describe research being done to develop commercially available perfusion systems to enhance organ preservation.

| S-EPMC6153619 | biostudies-literature

Brachylophosaurus In Gel Digestion

Project description:Protein extracts from the dinosaur Brachylophosaurus canadensis, as well as sediment and blank controls, were in-gel digested and analyzed by LC-MS-MS.

2017-01-24 | PXD005087 | Pride

The Effect of Gel Microstructure on Simulated Gastric Digestion of Protein Gels.

Project description:The objective of this study was to analyse the impact of the gel structure obtained by different heat-induced temperatures on the in vitro gastric digestibility at pH 2. To achieve this, gels were prepared from soy protein, pea protein, albumin from chicken egg white and whey protein isolate at varying temperatures (90, 120 and 140 °C) for 30 min. Gels were characterised prior to digestion via microstructure and SDS-PAGE analysis. Subsequently, the gastric digestion process was followed via the protein hydrolysis and HPSEC analysis up to 180 min. Peptides of different sizes (<5 kDa) were gradually formed during the digestion. Our results showed that gels induced at 140 °C were digested faster. The protein source and gelation temperature had great influence on the in vitro gastric protein digestibility.

| S-EPMC5937947 | biostudies-literature

Reversible gel-sol photoswitching with an overcrowded alkene-based bis-urea supergelator.

Project description:A new type of low-molecular-weight gelator (LMWG), i.e. overcrowded alkene-based bis-ureas, can be switched effectively between cis and trans isomers using light as demonstrated by 1H NMR and UV-Vis spectroscopy. Gelation studies reveal that one of the synthesized trans compounds forms stable gels in aromatic hydrocarbon solvents down to a critical concentration of 0.4 mg mL-1. Transmission electron microscopy (TEM) shows that this gel consists of an entangled fibrous network. For the trans isomer of this LMWG intermolecular urea hydrogen bonding is observed in the solid state, whereas density functional theory (DFT) geometry optimization of the cis isomer indicates the possible formation of an intramolecular hydrogen bond. Irradiation of the gel triggers trans-to-cis isomerization and consequently, a gel-sol phase transition. This process can be fully reversed by altering the irradiation wavelength.

| S-EPMC6013809 | biostudies-literature

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