Proceedings of Oncobiology Symposiums
Proceedings of XIV Oncobiology Symposium

INTRODUCTION AND OBJECTIVES: Tumor cells have an increased uptake of glucose. This behavior is known to be necessary to fulfill their goal of rapid growth and to supply their intense metabolic needs. This phenomenon has been widely observed in conditions close to normoglycemia (or low-glucose, LG, 5 mM) in vivo or in cells cultured at high glucose concentrations (HG, 25 mM) or unknown concentrations in vitro, but not much had been done to evaluate the difference between these two conditions, which is relevant for metabolic diseases such as diabetes, nor the effects of these different glucose concentration on the cell surface glycans. Previously we’ve shown that murine colon cancer cells (MC38) cultured in HG have a higher proliferation and invasion rate while presenting an abnormal cell surface glycosylation profile evaluated through a lectin’s array assay using flow cytometry. Our goal with this study is to provide an in-depth of the glycosylation of membrane proteins of colon cancer cells through mass spectrometry. MATERIAL AND METHODS: As glycans mass spectrometry has never been done within the facilities of the Centro de Espectrometria de Massas de Biomoléculas (CEMBIO), we first have to setup a working protocol. Briefly, MC38 cells were cultured in LG or HG and their membrane proteins were extracted by various methods, reduced, alkylated, digested by Trypsin and the N-Glycans were removed by PNGase or hydrazine using different workflows. These glycans were purified and tagged with procainamide and subjected to Matrix Assisted Laser Desorption Ionization Mass Spectrometry with a Time of Flight (MALDI-TOF) or Magnetic Resonance (MALDI-MRMS) detector or liquid chromatography associated with an Electrospray Ionization Mass Spectrometer (LC-MS). The data was analyzed using Bruker Compass Data Analysis and Glycoworkbench. RESULTS AND CONCLUSION: Protein extractions were done by different methods and whole cell lysates have been shown to be the more consistent and efficient method, when compared to freeze-thaw cell lysis for membrane purification or density gradient ultracentrifugation. Multiple methods were used to extract glycans, and PNGase F in-solution digestion provided a more efficient extraction when compared to in-membrane digestion or hydrazine in-solution chemical extraction. A novel method involving whole cells shaving of glycans has been successful, though only with non-adherent cells. With this, a valid workflow has been established and further experiments can be made in order to evaluate the differences between glycans from MC38 cells cultured in LG and HG. The novel method is promising, since it allows for further analysis of the “shaved” cells through commonly used tools such as flow cytometry, but needs to be further refined.