A comprehensive factorial design study of variables affecting protein extraction from formalin-fixed kidney tissue samples

Formalin-fixed tissues are an important source of biological samples for biomedical research. However, proteomics analysis of formalin-fixed tissues has been set aside by formalin-induced protein modifications, which reduce protein extraction efficiency. In this study, a two level full factorial exp...

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Bibliographic Details
Published in:Talanta (Oxford) 2014-02, Vol.119, p.90-97
Main Authors: Araújo, J.E., Oliveira, E., Otero-Glez, A., Santos Nores, J., Igrejas, G., Lodeiro, C., Capelo, J.L., Santos, H.M.
Format: Article
Language:English
Subjects:
Buffers
Chromatography, Liquid
Design engineering
Electrophoresis, Polyacrylamide Gel
Energy use
Extraction
Factorials
Formaldehyde
Formalin-fixed
Humans
Kidney - chemistry
Kidneys
Optimization
Protein extraction
Proteins
Proteins - isolation & purification
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Tandem Mass Spectrometry
Tissue proteomics
Ultrasonic
Ultrasound
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Summary:Formalin-fixed tissues are an important source of biological samples for biomedical research. However, proteomics analysis of formalin-fixed tissues has been set aside by formalin-induced protein modifications, which reduce protein extraction efficiency. In this study, a two level full factorial experimental design (24) was used to determine the effects of the extracting conditions in the efficiency of protein recovery from formalin-fixed kidney samples. The following variables were assessed: temperature of extraction, pH of extraction, composition of the extracting buffer and the use ultrasonic energy applied with probe. It is clearly demonstrated that when hating and ultrasonic energy are used in conjunction, a 7-fold increase (p < 0.05) in protein extraction is obtained if compared to extracting conditions for which neither heating nor ultrasonic energy are used. The optimization study was done following the amount of protein extracted by UV (Nanodrop® technology, protein ABS at 280nm) and by 1D SDS-PAGE. Extracts obtained with the optimized conditions were subjected to LC-MALDI MS/MS. A total of 112 proteins were identified. [Display omitted] •Two level full factorial experimental design (24) applied to tissue protein extraction.•Improved protein extraction protocol from formalin-fixed kidney tissue.•Using heating and ultrasonication, pH and glycerol do not influence the amount of total protein extracted or the amount of proteins identified.•Using optimum conditions a 7-fold increase (p
ISSN:0039-9140
1873-3573
DOI:10.1016/j.talanta.2013.10.019