Quantification, 2de analysis and identification of enriched glycosylated proteins from mouse muscles: difficulties and alternatives

Quantification, 2de analysis and identification of enriched glycosylated proteins from mouse muscles: difficulties and alternatives

Author Menegoci Eugenio, Patricia de Fatima Google Scholar
Assunção, Nilson Antonio Autor UNIFESP Google Scholar
Sciandra, Francesca Google Scholar
Aquino, Adriano Google Scholar
Brancaccio, Andrea Google Scholar
Carrilho, Emanuel Google Scholar
Abstract One of the problems with 2DE is that proteins present in low amounts in a sample are usually not detected, since their signals are masked by the predominant proteins. The elimination of these abundant proteins is not a guaranteed solution to achieve the desired results. The main objective of this study was the comparison of common and simple methodologies employed for 2DE analysis followed by MS identification, focusing on a pre-purified sample using a wheat germ agglutinin (WGA) column. Adult male C57Black/Crj6 (C57BL/6) mice were chosen as the model animal in this study

the gastrocnemius muscles were collected and processed for the experiments. The initial fractionation with succinylated WGA was successful for the elimination of the most abundant proteins. Two quantification methods were employed for the purified samples, and bicinchoninic acid (BCA) was proven to be most reliable for the quantification of glycoproteins. The gel staining method, however, was found to be decisive for the detection of specific proteins, since their structures affect the interaction of the dye with the peptide backbone. The Coomassie Blue R-250 dye very weakly stained the gel with the WGA purified sample. When the same gel was stained with silver nitrate, however, MS could positively assign 12 new spots. The structure of the referred proteins was not found to be prone to interaction with Coomassie blue.
Keywords Gel-Staining Methods
Glycoprotein
Protein Quantification
Two-Dimensional Gel ElectrophoresisLectin Affinity-Chromatography
2-Dimensional Gel-Electrophoresis
Coomassie Brilliant Blue
Wheat-Germ-Agglutinin
Mass-Spectrometry
Membrane-Glycoproteins
Muscular-Dystrophies
Biomarker Discovery
Polyacrylamide-Gels
Bicinchoninic Acid
Language English
Sponsor FAPESP [2008/04050-4, 2009/54040-8, 2009/16598-7]
CNPq [140840/2008-7, 160528/2011-9]
CAPES [0203-10-6]
Grant number FAPESP: 2008/04050-4
FAPESP: 2009/54040-8
FAPESP: 2009/16598-7
CNPq: 140840/2008-7
CNPq: 160528/2011-9
CAPES: 0203-10-6
Date 2016
Published in Electrophoresis. Hoboken, v. 37, n. 2, p. 321-334, 2016.
ISSN 0173-0835 (Sherpa/Romeo, impact factor)
Publisher Wiley-blackwell
Extent 321-334
Origin https://doi.org/10.1002/elps.201500362
Access rights Closed access
Type Article
Web of Science ID WOS:000368030000011
URI http://repositorio.unifesp.br/handle/11600/49657

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