doi:10.1016/j.jchromb.2004.01.019
Copyright © 2004 Elsevier B.V. All rights reserved.
Concanavalin A chromatography coupled to two-dimensional gel electrophoresis improves protein expression studies of the serum proteome
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Ana María Rodríguez-Piñeiro a, Daniel Ayude a, b, Francisco Javier Rodríguez-Berrocal a and María Páez de la Cadena 
, 
, a
a Departamento de Bioquímica, Genética e Inmunología, Facultad de Ciencias, As Lagoas Marcosende, s/n 36200, Vigo, Spain
b Lema y Bandín Laboratorios, Lepanto 5, 36204, Vigo, Spain
Received 8 August 2003;
Revised 23 December 2003;
accepted 12 January 2004.
Available online 8 February 2004.
Abstract
In the present study, we show a simple method to analyse human serum proteins using Concanavalin A (Con A) chromatography coupled to two-dimensional gel electrophoresis. Serum samples were separated into two fractions, one mainly containing non-glycosylated and O-glycosylated proteins and the other enriched in N-glycosylated proteins. Both fractions were subjected to two-dimensional gel electrophoresis, and the obtained maps were analysed. The method presented here improves the resolution of the serum proteome, increasing the number of visualized spots over two times and allowing the detection of proteins with lower abundance in serum. We have proved the feasibility of the method comparing the N-glycoprotein fraction of serum from donors and colorectal cancer (CRC) patients.
Author Keywords: Author Keywords: Glycosylation; Concanavalin A; Albumin
Fig. 1. (A) Representative profile of a Con A chromatography of serum. One millilitre of human serum was applied to a 5.5 ml column of Con A-Sepharose. Peak I: proteins mainly non-glycosylated and O-glycosylated. Peak II: fraction enriched in N-glycoproteins. (B) Median, mean ± standard deviation and percentage of the protein quantity measured in eight samples from healthy donors and five from colorectal cancer patients, and in Peaks I and II after Con A chromatography. α-mm: methyl α--mannopyranoside.
Fig. 2. One-dimensional SDS-PAGE of a donor total serum (Lane 1), chromatographic Fraction I (Lane 2) and Fraction II (Lane 3). Twenty micrograms of protein were electrophoresed at 90 V (4%T stacking gel) until the samples entered the 12% polyacrylamide gel, and then at 200 V until the bromophenol blue marker reached the bottom of the gel. LMW: low molecular weight markers.
Fig. 3. Representative silver-stained 2D-PAGE of total serum and Con A chromatographic Fractions I (mainly non-glycoproteins and O-glycoproteins) and II (enriched in N-glycoproteins). In these 2D-gels, we detected 1376 spots in serum, 841 in Fraction I and 1185 in Fraction II. Some major proteins are highlighted in all the three maps. Boxes 1, 2 and 3 are enlarged in Fig. 4. Mr: relative molecular mass; pI: isoelectric point; AACT: α-1-antichymotrypsin; A2HS: α-2-HS-glycoprotein; LRG: leucine-rich α-2-glycoprotein; Ig: immunoglobulin; ApoA-I: apolipoprotein A-I; RPB: plasma retinol-binding protein.
Fig. 4. Enlargement of Boxes 1, 2 and 3 (see Fig. 3 for reference) from representative 2D-maps of serum and chromatographic Fractions I and II. Spots highlighted correspond to: proteins not visualized in a total serum map, visible in Peak I, Peak II or both, depending on the glycosylation pattern (circles); spots increased over two-fold (arrows); and areas where better separation was achieved (rectangles). Box 1 accounts for 91 spots in total serum, 50 spots in Peak I and 113 in Peak II. Box 2 presents 105 spots in total serum, 43 in Peak I and 121 in Peak II. Box 3 shows 89 spots in total serum, 75 in Peak I and 105 in Peak II.
Table 1. N-Glycoproteins increased in colorectal cancer patients in comparison with healthy donors
SSP, standard spot number; n.s., not significative.
Table 2. N-Glycoproteins decreased in colorectal cancer patients in comparison with healthy donors
SSP, standard spot number, n.s., not significative.
Corresponding author. Fax: +34-986-812556.
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