Abundant plasma protein depletion using ammonium sulfate precipitation and Protein A affinity chromatography.
J Chromatogr B Analyt Technol Biomed Life Sci
; 1089: 43-59, 2018 Jul 01.
Article
em En
| MEDLINE
| ID: mdl-29758408
ABSTRACT
Plasma is a highly valuable resource for biomarker research since it is easy obtainable and contains a high amount of information on patient health status. Although advancements in the field of proteomics enabled analysis of the plasma proteome, identification of low abundant proteins remains challenging due to high complexity and large dynamic range. In order to reduce the dynamic range of protein concentrations, a tandem depletion technique consisting of ammonium sulfate precipitation and Protein A affinity chromatography was developed. Using this method, 50% of albumin, together with other high abundant proteins such as alpha-1-antitrypsin, was depleted from the plasma sample at 20% to 40% ammonium sulfate saturation levels. In combination with immunoglobulin removal using a Protein A column, this technique delivered up to 40 new low- to medium abundance protein identifications when performing a shotgun mass spectrometry analysis. Compared to non-depleted plasma, 270 additional protein spots were observed during 2D-PAGE analysis. These results illustrate that this tandem depletion method is equivalent to commercial kits which are based on immune-affinity chromatography. Moreover, this method using Protein A immunoglobulin depletion was shown to be highly reproducible and a minimal amount of non-target proteins was depleted. The combination of ammonium sulfate precipitation and Protein A affinity chromatography offers a low cost, efficient, straightforward and reproducible alternative to commercial kits, with proteins remaining in native conformation, allowing protein activity and protein interaction studies.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Precipitação Química
/
Proteína Estafilocócica A
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Proteínas Sanguíneas
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Cromatografia de Afinidade
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Sulfato de Amônio
Tipo de estudo:
Prognostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article