ORIGINAL RESEARCH: Comparison of methods for depletion of albumin and IgG from equine serum

Olver, Christine S.; Webb, Teckla L.; Long, Lindsey J.; Scherman, Hataichanok; Prenni, Jessica E.

doi:10.1111/j.1939-165x.2010.00241.x

Cited by 15 publications

(9 citation statements)

References 16 publications

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“…Using crude plasma for 2-DE leads to overloading of abundant proteins and poor spot resolution. Thus, albumin removal is a crucial preparation step for plasma proteome investigation with in-gel methods (Olver et al 2010). In present work we tested four different albumin removal strategies for in-gel proteomic approaches.…”

Section: Discussionmentioning

confidence: 99%

A comparison of albumin removal procedures for proteomic analysis of blood plasma

Tomašcová

Lehotský²,

Kalenská³

et al. 2019

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Blood biomarkers are usually present in low concentration and can be masked by the high-abundance proteins, of which albumin is the predominant one. The purpose of this study was to compare four different albumin removal methods compatible with in-gel based proteomics, applicable for plasma, without requiring specific techniques and high financial input. Plasma underwent albumin depletion with ultrafiltration device Amicon Ultra, commercial ProteoPrep Blue Albumin and IgG Depletion Kit, acetonitrile precipitation method and precipitation with acetonitrile-methanol protocol. All samples were evaluated by 1-D and 2-D gel electrophoresis with subsequent mass spectrometry protein identification. Two of the tested methods (ProteoPrep BlueKit and acetonitrilemethanol precipitation) maintained sufficient protein content for further in-gel analyses. Their 2-D protein profiles were distinctively separated and overlapped with protein profile of crude plasma. Protein spot count showed significant increase in protein spots, compared to crude plasma, only with acetonitrile-methanol precipitation method. Precipitation with acetonitrile-methanol method significantly increased number of protein spots on 2-D protein profile and improved score of mass spectrometry identification. However, albumin was still present and found in number of protein spots.

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Section: Discussionmentioning

confidence: 99%

A comparison of albumin removal procedures for proteomic analysis of blood plasma

Tomašcová

Lehotský²,

Kalenská³

et al. 2019

gpb

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“…This is consistent with the results of Chen et al [ 3 ], who reported that 5–10% TCA in acetone is the optimal condition for removing human albumin and IgG, but Liu et al [ 14 ] reported that 1% TCA in isopropanol is the optimal solution to remove albumin from human and monkey serum for a proteomic study. Notably, our protocol is simple and inexpensive, and it is more efficient than using human commercial kits to remove equine serum albumin and IgG [ 22 ].…”

Section: Discussionmentioning

confidence: 99%

“…In human analyses, serum/plasma albumin is usually depleted by using column immunoaffinity purification or commercial albumin removal kits [ 30 ]. Unfortunately, there is no commercial kit available specifically for horses, and most kits for human samples inefficiently remove equine albumin [ 22 ]. Additionally, there is also a lack of published reports relating to the protocol of serum albumin removal in horses.…”

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confidence: 99%

Optimisation of a serum albumin removal protocol for use in a proteomic study to identify the protein biomarkers for silent gastric ulceration in horses

Poltep

Tesena

Yingchutrakul

et al. 2018

JES

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Silent gastric ulceration occurs without evidence of clinical signs and is common in horses. There is currently no a simple and effective method to diagnose this disease. Proteomics can be used to identify serum biomarkers, but the most abundant serum protein, albumin, could conceal candidate biomarkers. Therefore, it is recommended to remove albumin before a proteomic study; however, there is no specific albumin depletion kit or standard protocol available for horse samples. The objectives of this study were to optimise a protocol to remove equine serum albumin and to use albumin-depleted serum to identify the protein biomarkers for silent gastric ulceration. Gastroscopy was used to identify gastric ulceration, and serum was obtained from horses with either a healthy gastric mucosa or gastric ulceration. Serum albumin was removed using the trichloroacetic acid (TCA) protein precipitation method, and this protocol was optimised by varying the concentration of TCA, type of organic solvents, ratio of serum to protein precipitation solution, and incubation times. Electrophoresis and image analysis were used to compare the amounts of albumin, immunoglobulins G (IgG), and protein degradation before and after TCA precipitation. The best protocol was chosen to remove albumin for a proteomic study (electrophoresis and mass spectrometry). The results revealed that protocol 2 (ratio of serum to solution 1:5, 10% TCA in acetone, and 90 min incubation) was the most efficient protocol to remove albumin (98%) and IgG heavy (80%) and light (98%) chains without degrading other proteins. After electrophoresis and mass spectrometry analysis, KRT1, KRT6A and KRT18 were identified as potential markers for silent gastric ulceration.

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“…Serum albumin is synthesized mainly in the liver (1) and secreted into blood where it constitutes almost 50% of the proteins in the serum (2–5). Albumin also produces the majority of the protein contents of extracellular fluids such as interstitial, cerebrospinal, and lymphatic (2,6,7).…”

Section: Introductionmentioning

confidence: 99%

Isolation and characterization of serum albumin from Camelus dromedarius

Malik

Al-Senaidy

Skrzypczak‐Jankun

et al. 2013

Experimental and Therapeutic Medicine

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Serum albumin constitutes 35–50 mg/ml of plasma proteins and performs various physiological activities including the regulation of osmotic pressure on blood, maintaining buffering of the blood pH, carrying different fatty acids and other small molecules, such as bilirubin, hormones, drugs and metal ions, as well as participating in immunological responses. Serum albumin is an extensively used protein in biotechnological and pharmaceutical industries. The camel (Camelus dromedarius) is well tailored to successfully survive in extremely hot and dry climates. Plasma osmolality in the camel increases during water-deprived conditions. In such circumstances serum albumin is crucial in the regulation of blood pressure. The study of biochemical, biophysical and immunological aspects of camel serum albumin (CSA) are likely to provide molecular insights into camel physiology and may render it an alternative to human serum albumin (HSA) and bovine serum albumin (BSA) in all cases. However, these proteins are currently not available or cannot be utilized due to a variety of considerations. In this study, 12 mg of highly pure CSA was obtained from 1 ml plasma. Coomassie Brilliant Blue staining of SDS-PAGE yielded one band and RP-HPLC results revealed a single sharp peak, indicating homogenous preparation of the CSA. The charge/mass ratio and surface hydrophobicity of the CSA was similar to that of BSA. Mass spectrometry analysis of the purified protein confirmed the identity of CSA.

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ORIGINAL RESEARCH: Comparison of methods for depletion of albumin and IgG from equine serum

Abstract: Albumin and IgG removal kits optimized for human use have variable efficacy for equine serum. Combined use of the ProteoExtract kit and manual incubation with protein G Sepharose beads provided the most effective depletion.

Cited by 15 publications

References 16 publications

A comparison of albumin removal procedures for proteomic analysis of blood plasma

A comparison of albumin removal procedures for proteomic analysis of blood plasma

Optimisation of a serum albumin removal protocol for use in a proteomic study to identify the protein biomarkers for silent gastric ulceration in horses

Isolation and characterization of serum albumin from Camelus dromedarius

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