Quantification, 2DE analysis and identification of enriched glycosylated proteins from mouse muscles: Difficulties and alternatives

Author(s)

Menegoci Eugenio, Patricia de Fatima ^{[1, 2]} ; Assuncao, Nilson Antonio ^[3] ; Sciandra, Francesca ^[4] ; Aquino, Adriano ^{[1, 2]} ; Brancaccio, Andrea ^[4] ; Carrilho, Emanuel ^{[1, 2]}

Affiliation

^[1] Univ Sao Paulo, Inst Quim Sao Carlos, Sao Carlos, SP - Brazil

^[2] Inst Nacl Ciencia & Tecnol Bioanalit, Campinas, SP - Brazil

^[3] Univ Fed Sao Paulo, Inst Ciencias Ambientais Quim & Farmaceut, Diadema, SP - Brazil

^[4] Univ Cattolica Sacro Cuore, Ist Biochim & Biochim Clin, Ist Chim Riconoscimento Mol, CNR, Rome - Italy

• Document type: Journal article
• Source: ELECTROPHORESIS; v. 37, n. 2, p. 321-334, JAN 2016.
• Web of Science Citations: 0

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. (AU)

• FAPESP's process: 09/54040-8 - Acquisition of a high-resolution Orbitrap mass spectrometer for the discovery and structural analysis of biologically active compounds: applications in proteomics, biomarkers, synthesis, isolation, and characterization of natural products, studies of redox systems in food and enzymatic synthesis
• Grantee: Emanuel Carrilho
• Support type: Multi-user Equipment Program
• FAPESP's process: 08/04050-4 - Bidimensional electrophoresis as a fundamental tool in proteomics analysis
• Grantee: Emanuel Carrilho
• Support type: Regular Research Grants
• FAPESP's process: 09/16598-7 - Glycomic study of alpha-dystroglycan and of the glycoproteic profile of dystrophic animal models
• Grantee: Emanuel Carrilho
• Support type: Regular Research Grants