title: Quantification, 2DE analysis and identification of enriched glycosylated proteins from mouse muscles: Difficulties and alternatives.
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.
authors.affiliation: Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil.|Instituto Nacional de Ciência e Tecnologia de Bioanalítica, Campinas, SP, Brazil.
authors.forename: Patrícia
authors.initials: P
authors.lastname: de Fátima MenegociEugênio
authors.affiliation: Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo, Diadema, SP, Brazil.
authors.forename: Nilson Antonio
authors.initials: NA
authors.lastname: Assunção
authors.affiliation: Istituto di Chimica del Riconoscimento Molecolare (CNR), c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Roma, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Sciandra
authors.affiliation: Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil.|Instituto Nacional de Ciência e Tecnologia de Bioanalítica, Campinas, SP, Brazil.
authors.forename: Adriano
authors.initials: A
authors.lastname: Aquino
authors.affiliation: Istituto di Chimica del Riconoscimento Molecolare (CNR), c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Roma, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Brancaccio
authors.affiliation: Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil.|Instituto Nacional de Ciência e Tecnologia de Bioanalítica, Campinas, SP, Brazil.
authors.forename: Emanuel
authors.initials: E
authors.lastname: Carrilho
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D004396:Coloring Agents / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis* / Q000302:isolation & purification; D006031:Glycosylation; D008297:Male; D008810:Mice, Inbred C57BL; D009124:Muscle Proteins / Q000032:analysis / Q000302:isolation & purification; D009132:Muscles / Q000737:chemistry*; D012394:Rosaniline Dyes / Q000032:analysis; D016622:Silver Staining / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D014909:Wheat Germ Agglutinins / Q000737:chemistry
chemical_list: D004396:Coloring Agents; D006023:Glycoproteins; D009124:Muscle Proteins; D012394:Rosaniline Dyes; D014909:Wheat Germ Agglutinins; C030717:succinylated wheat germ agglutinin; C024757:Coomassie brilliant blue R; C048139:Coomassie blue
keywords: Gel-staining methods; Glycoprotein; Protein quantification; Two-dimensional gel electrophoresis