Combining results from lectin affinity chromatography and glycocapture approaches substantially improves the coverage of the glycoproteome

Abstract

Identification of glycosylated proteins, especially those in the plasma membrane, has the potential of defining diagnostic biomarkers and therapeutic targets as well as increasing our understanding of changes occurring in the glycoproteome during normal differentiation and disease processes. Although many cellular proteins are glycosylated they are rarely identified by mass spectrometric analysis (e.g. shotgun proteomics) of total cell lysates. Therefore, methods that specifically target glycoproteins are necessary to facilitate their isolation from total cell lysates prior to their identification by mass spectrometry-based analysis. To enrich for plasma membrane glycoproteins the methods must selectively target characteristics associated with proteins within this compartment. We demonstrate that the application of two methods, one that uses periodate to label glycoproteins of intact cells and a hydrazide resin to capture the labeled glycoproteins and another that targets glycoproteins with sialic acid residues using lectin affinity chromatography, in conjunction with liquid chromatography-tandem mass spectrometry is effective for plasma membrane glycoprotein identification. We demonstrate that this combination of methods dramatically increases coverage of the plasma membrane proteome (more than one-half of the membrane glycoproteins were identified by the two methods uniquely) and also results in the identification of a large number of secreted glycoproteins. Our approach avoids the need for subcellular fractionation and utilizes a simple detergent lysis step that effectively solubilizes membrane glycoproteins. The plasma membrane localization of a subset of proteins identified was validated, and the dynamics of their expression in HeLa cells was evaluated during the cell cycle. Results obtained from the cell cycle studies demonstrate that plasma membrane protein expression can change up to 4-fold as cells transit the cell cycle and demonstrate the need to consider such changes when carrying out quantitative proteomics comparison of cell lines.

Fig. 1.

Work flow diagram of the periodate oxidation/hydrazide resin and the M. amurensis chromatography methods.

Fig. 2.

Subcellular location of glycoproteins identified in HeLa cells by the periodate/hydrazide method. RER, rough ER.

Fig. 3.

MS/MS spectrum of the N-linked tryptic peptide containing amino acids 316–328 from CD98. Asn-323 has been converted to Asp by PNGase F as shown by the mass difference of 115 Da between fragments y5 and y6.

Fig. 4.

Subcellular location of glycoproteins detected in the PNGase F-treated, tryptic digests of HeLa cell proteins bound to M. amurensis resin. RER, rough ER.

Fig. 5.

Western blot of CD antigens in HeLa cell lysates. HeLa cell extracts (5 and 10 μg) were separated on 4–12% bis-Tris SDS-polyacrylamide gels using non-reducing (top) or reducing (CD147 blot not shown) conditions (bottom). Bands indicated by the brackets were specifically recognized by anti-CD antibodies.

Fig. 6.

Antibody binding curves to HeLa cell lysates and CD antigen expression in HeLa cells during various parts of the cell cycle (A–D). Left panels, dot blot and corresponding signal intensity plots for the binding of CD antibodies to HeLa lysates (1.7–4,000 ng) prepared from cells grown asynchronously. The lysate was dotted (2-fold dilutions), probed for the corresponding CD antigens, and detected using ECL Plus chemifluorescence (corresponding dot blot shown above each graph). Right panels, dot blots of HeLa lysates prepared from synchronized cells collected every 2 h after release from a double thymidine block. The dot blots (not shown) were probed for CD antigens, and the bound antibody was detected using ECL chemifluorescence. Relative intensities were an average of three sets of normalized data using two amounts of lysate in duplicate. Maximum error in any measurement was 18.5%.