Abstract 4642: Ion mobility separation of N-Glycans directly from ffpe colon cancer tissue section in a MALDI imaging experiment
Authors: Claude Emmanuelle , Angel Peggi M. , Drake Richard R. , Olivos Hernando , Langridge James I. 
Published: July 2017

Colon cancer is currently the third most common cancer in both men and women. Research studies have reported extensive alterations in protein glycosylation patterns in cancer tissues. However during these studies, the tissues are homogenized and the spatial information showing the localization of the glycans is lost. Mass spectrometry imaging (MSI) is an established analytical tool for biomolecular research which can accurately determine the spatial location of molecules in a tissue section. Recently, methods have been developed to determine released N-Glycans directly from tissues. A major challenge in the analysis of N-glycans is the large number of isobaric glycans resulting from their complex structures with branched chains and multiple additions residues. Here we report the advantage of ion mobility separation to differentiate these glycans in a MALDI MSI workflow used in the analysis of human FFPE colon cancer tissue. 5µm FFPE tissue were were deparaffinized in xylene, rehydrated in different composition of ethanol/water solutions, transferred in citraconic anhydride buffer for antigen retrieval and water washed. To release the glycans from their proteins, a PNGaseF solution was sprayed on the tissue. A solution of MALDI was sprayed onto the tissue. Experiments were carried out on a SYNAPT G2-Si HDMS system(Waters) where the tri-wave separated ions according to their ion mobility in the gas phase. The overall MALDI spectrum shows strong signal for N-glycan molecules, demonstrating the efficacy of the digestion step of the methodology. Using prior knowledge of the type of glycans, 76 glycans were identified and mapped directly from the FFPE tissue section, from a mass to charge ratio (m/z) of 771.5 up to 2905.03 using accurate mass information. MALDI MSI was able to distinguish the tissue morphology and determine the tumor region based on specific ions, especially sodiated N-Glycans Hex7HexNAc2 (m/z 1581.5) which was highly abundant in the tumor tissue. When the IMS was explored using the DriftScope software, it could clearly been observed that two nested trendlines in m/z vs. DriftTime existed. The faster trendline corresponding to more compact conformation of the ions in the gas phase was identified to be the N-Glycan class of molecules. Using IMS the MS data showed a clear picture of the N-Glycans compare to the MS data alone. In several cases, the IMS peaks were broader than the expected resolution or there were shoulder peaks, indicating that isobaric species were present. For example, m/z 1444.6 ( Hex4dHex1HexNAc3,Na+) displayed two IMS distinct peaks which while not baseline separated were sufficiently separated to obtain individual ion images which showed significantly different distributions. The isobaric species with the faster drift time was evenly distributed across the tissue type of the section, whereas the isobaric species that had the slower drift time was less abundant in the cancer tissue.

Citation Format: Emmanuelle Claude, Peggi M. Angel, Richard R. Drake, Hernando Olivos, James I. Langridge. Ion mobility separation of N-Glycans directly from ffpe colon cancer tissue section in a MALDI imaging experiment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4642. doi:10.1158/1538-7445.AM2017-4642