Comparative glycoproteomics based on lectins affinity capture of N‐linked glycoproteins from human Chang liver cells and MHCC97‐H cells

Abstract

We present here an effective technique for the large-scale separation and identification of N-linked glycoproteins from Chang liver cells, the human normal liver cells. To enrich N-linked glycoproteins from the whole cells, a procedure containing the lysis of human liver cells, the solubilization of total proteins, lectin affinity chromatography including Concanavalin A and wheat germ agglutinin was established. Furthermore, captured N-linked glycoproteins were separated by 2-DE, and identified by MS and database searching. Finally, we found 63 N-glycoproteins in Chang liver cells. In addition, using the above method, we identified 7 remarkably up-regulated glycoproteins from MHCC97-H cells, highly metastatic liver cancer cells, compared to Chang liver cells. These up-regulated glycoproteins were associated with liver cancer and might be used as biomarkers for tumor diagnosis. Results showed that we established a high-throughput proteomic analysis for separating N-linked glycoproteins from human liver cells. This strategy greatly improved the glycoprotein analysis method associated with proteome-wide glycosylation changes related to liver cancer. Our work was part of the HUPO Human Liver Proteome Project (HLPP) studies and was supported by CHINA HUPO.

Up-regulation of prohibitin 1 is involved in the proliferation and migration of liver cancer cells

“…WGA affinity chromatography, 2-DE and MS analysis were performed as previously described [10]. Briefly, agarosebound WGA (Vector Laboratories, Al-1023) was prepared and used to capture glycoproteins from the soluble fractions of Chang liver and MHCC97-H lysates.…”

“…The eluted fractions were dialyzed with Milli Q water (Millipore, USA) at 4°C and freeze-dried. 2-DE was performed as previously described [10] using the IPGphor IEF System and a Hoefer SE 600 (Amersham Biosciences, Sweden). Glycoproteins (250 μg) were assessed by isoelectric focusing (IEF) using 13 cm pH 3-10 NL IPG strips (Amersham Biosciences).…”

“…Using two-dimensional electrophoresis (2-DE) and mass spectrometry (MS), we separated and identified five markedly up-regulated glycoproteins from MHCC97-H cells (published in the journal of Proteomics) [10]. Of these, we focused on a new glycoprotein, PHB.…”

Glycoproteomic Reactor for Human Plasma

“…Alternatively, Xu et al dialyzed the eluted fraction from ConA column using Milli Q water at 4°C to remove the R-Dmannopyranoside. 24 As well, a repeated buffer exchange during repeated ultrafiltration has been performed to remove R-mannopyranoside by Lewandrowski et al 32 However, dialysis, ultrafiltration and TCA, or acetone precipitations are all multistep and time-consuming processes which result in sample variation, loss of sample, contamination, and so forth. We compared the performance of the glycoproteomic reactor for capturing glycoproteins in the presence of R-D-mannopyranoside to the standard TCA/acetone precipitation for removing R-mannopyranoside.…”

“…21 Immobilized lectins have been extensively used for glycoproteomic research for the capture of glycoproteins. 9,17,23,24 The most common lectin method uses Concavalin A (ConA), which binds very tightly to high-mannose-type N-glycans. 25 ConA affinity chromatography has been used in human plasma, but to date, few glycopeptides or glycosites have been identified.…”