Unlike other high molecular weight biomolecules, polysaccharides are extremely heterogeneous in terms of size, structure and chemical functions, posing a clear challenge to the field of analytical chemistry. Mass spectrometry (MS), with its remarkable sensitivity, speed and high information content, is a forefront method for deciphering biopolymers. However, classical MS approach fails in many cases to achieve definitive structural assignments of carbohydrates. The methodology commonly used for MS based approach involved digesting polymers, using specific enzymes, into a complex mixture of oligosaccharides, which include numerous isoforms. With this approach, in order to avoid the ubiquity of small major structures, the challenge is to obtain the largest possible oligosaccharides while developing efficient separation techniques compatible with these molecule sizes and approaches for their structural characterizations. Our work explores the use and development of two innovative MS-based approaches for characterizing the fine structure of carbohydrates derived from plant and algal cell walls. The first use a new generation ion mobility (IM)-MS that allows provide unprecedented resolutions on all stages of fragmentation using a cyclic cell [1]. We introduce notably the concept of IMSn sequencing [2,3] and molecular networking to preprocess the data [4]. The second is based on recently introduced methods using high-energy activation pathway [5-7] called charge transfer dissociation (CTD) and electron activated dissociation (ExD) that lead to a variety of diagnostic fragments to solve isomerism questions based on the positioning of modifications and the branching pattern between subunits. Our results illustrate the potential of these emerging techniques to push back the existing analytical barriers in the field of structural chemistry of carbohydrates. References [1] D Ropartz, M Fanuel, J Ujma, M Palmer, K Giles, H Rogniaux, Anal. Chem. 91 (18), 12030 (2019) [2] S Ollivier, et al. Anal. Chem. 93 (15), 6254 (2021) [3] S Ollivier, D Ropartz, M Fanuel, H Rogniaux, H. Anal. Chem. 95 (26), 10087 (2023) [4] S Ollivier, M Fanuel, H Rogniaux, D Ropartz, Anal. Chem. 93 (31), 10871 (2021) [5] D Ropartz, P Li, GP Jackson, H Rogniaux. Anal. Chem. 89 (7), 3824 (2017) [6] H Buck-Wiese, et al. J. Am. Soc. Mass Spectrom. 31 (6), 1249 (2020) [7] D Ropartz, et al. Anal. Chem. 94 (4), 2279 (2022)