Hydrophilic interaction chromatography of intact, soluble proteins.
Abstract: The separation of intact proteins by means of Hydrophilic Interaction Chromatography (HILIC) was demonstrated with human apoA-I, recombinant human apoM, and equine cytochrome C. Five different commercially available HILIC columns were compared. Using one of these columns, different glycosylated isoforms of apoM were separated from each other and from the aglyco-form.
Copyright © 2010 Elsevier B.V. All rights reserved.
Publication Date: 2010-09-17 PubMed ID: 20926084DOI: 10.1016/j.chroma.2010.09.027Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research article explores the use of Hydrophilic Interaction Chromatography (HILIC) in separating intact soluble proteins, testing five commercially available HILIC columns, using human apoA-I, apoM, and equine cytochrome C for demonstration.
HILIC method for protein separation
- The research study employed Hydrophilic Interaction Chromatography (HILIC), which is a separation method for proteins. This is particularly useful in the analysis of intact, soluble proteins, such as those used in this study – human apoA-I, human apoM, and equine cytochrome C.
- HILIC works on the principle that polar molecules get retained on the polar stationary phase, allowing the separation of complex mixtures in a liquid formulation. The separation is due to the differential interaction of the proteins with the stationary phase.
Comparison of HILIC columns
- Five commercially available HILIC columns were evaluated in the study as part of the methodology to determine the most appropriate equipment for protein separation.
- The comparisons would have likely involved measuring performances such as speed, efficiency, resolution, and capacity in separating protein isoforms.
Separation of protein isoforms
- The applicability of the method is demonstrated by the successful separation of different glycosylation isoforms of apoM from each other and from the aglyco-form.
- Glycosylation isoforms are different forms of a protein that occur due to variations in the attachment of carbohydrate (sugar) groups, and being able to separate these is crucial for detailed protein characterization.
- The aglyco-form of a protein lacks a carbohydrate group, and once again, the separation achieved in this study and the identification of discrete peaks would illustrate the effectiveness of the HILIC method in isolating individual protein forms, a crucial task in many fields including pharmaceuticals and biomedicine.
Cite This Article
APA
Tetaz T, Detzner S, Friedlein A, Molitor B, Mary JL.
(2010).
Hydrophilic interaction chromatography of intact, soluble proteins.
J Chromatogr A, 1218(35), 5892-5896.
https://doi.org/10.1016/j.chroma.2010.09.027 Publication
Researcher Affiliations
- F. Hoffmann-La Roche Ltd, Discovery Technologies, PROBMX21, Basel, Switzerland. tim.tetaz@roche.com
MeSH Terms
- Animals
- Apolipoprotein A-I / chemistry
- Apolipoprotein A-I / isolation & purification
- Apolipoproteins / chemistry
- Apolipoproteins / isolation & purification
- Apolipoproteins M
- Cell Line, Tumor
- Chromatography, Liquid / instrumentation
- Chromatography, Liquid / methods
- Cytochromes c / chemistry
- Cytochromes c / isolation & purification
- Horses
- Humans
- Hydrophobic and Hydrophilic Interactions
- Lipocalins / chemistry
- Lipocalins / isolation & purification
- Protein Isoforms
- Proteins / chemistry
- Proteins / isolation & purification
- Solubility
Citations
This article has been cited 9 times.- Passamonti M, de Roos C, Schoenmakers PJ, Gargano AFG. Poly(acrylamide-co-N,N'-methylenebisacrylamide) Monoliths for High-Peak-Capacity Hydrophilic-Interaction Chromatography-High-Resolution Mass Spectrometry of Intact Proteins at Low Trifluoroacetic Acid Content.. Anal Chem 2021 Dec 7;93(48):16000-16007.
- Bupp CR, Schwartz C, Wei B, Wirth MJ. Protein-induced conformational change in glycans decreases the resolution of glycoproteins in hydrophilic interaction liquid chromatography.. J Sep Sci 2021 Apr;44(8):1581-1591.
- Gargano AFG, Roca LS, Fellers RT, Bocxe M, Domínguez-Vega E, Somsen GW. Capillary HILIC-MS: A New Tool for Sensitive Top-Down Proteomics.. Anal Chem 2018 Jun 5;90(11):6601-6609.
- Astefanei A, Dapic I, Camenzuli M. Different Stationary Phase Selectivities and Morphologies for Intact Protein Separations.. Chromatographia 2017;80(5):665-687.
- Tengattini S, Domínguez-Vega E, Temporini C, Terreni M, Somsen GW. Monitoring antigenic protein integrity during glycoconjugate vaccine synthesis using capillary electrophoresis-mass spectrometry.. Anal Bioanal Chem 2016 Sep;408(22):6123-32.
- Chen B, Peng Y, Valeja SG, Xiu L, Alpert AJ, Ge Y. Online Hydrophobic Interaction Chromatography-Mass Spectrometry for Top-Down Proteomics.. Anal Chem 2016 Feb 2;88(3):1885-91.
- Sentkowska A, Biesaga M, Pyrzynska K. Retention Study of Flavonoids Under Different Chromatographic Modes.. J Chromatogr Sci 2016 Apr;54(4):516-22.
- Pedrali A, Tengattini S, Marrubini G, Bavaro T, Hemström P, Massolini G, Terreni M, Temporini C. Characterization of intact neo-glycoproteins by hydrophilic interaction liquid chromatography.. Molecules 2014 Jun 30;19(7):9070-88.
- Zhang Z, Wu Z, Wirth MJ. Polyacrylamide brush layer for hydrophilic interaction liquid chromatography of intact glycoproteins.. J Chromatogr A 2013 Aug 2;1301:156-61.
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