Protocols for Assessing Storage Stability and Solubility of Released N-Linked Glycans Prior to Mass Spectrometry Analysis

Released N-linked glycans are frequently stored between enzymatic release, cleanup, and LC-MS analysis, yet storage solvent and freeze-thaw handling are often reported incompletely or omitted. This unit provides a procedure for determining, within a given laboratory and for a given glycan preparation, whether a selected storage condition preserves analytical performance prior to LC-ion mobility-mass spectrometry (LC-IM-MS). The workflow compares matched aliquots prepared from a single cleared glycan stock, using extracted ion chromatogram signal intensity, chromatographic peak shape, visible precipitation, and replicate precision as acceptance criteria, assessed both before and after a controlled freeze-thaw cycle. A worked example comparing 0.1% (v/v) formic acid in water with 50:50 acetonitrile:water is presented to demonstrate the workflow. The aqueous condition retained LC-IM-MS signal after one freeze-thaw cycle, while the acetonitrile-containing condition showed substantial post-thaw signal loss despite acceptable pre-freeze performance. This example illustrates the magnitude of effect that storage solvent can produce and is not presented as a general storage recommendation, since susceptibility is both structure dependent and platform dependent. The protocols are intended for laboratories analyzing released N-linked glycans from glycoproteins or biological samples and can be applied to PGC-, reversed-phase-, or HILIC-based LC-MS workflows, with solvent adjustment performed immediately before injection rather than during long-term storage. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Solvent selection and solubility assessment of released N-linked glycans before LC-IM-MS analysis Basic Protocol 2: Freeze-thaw stability evaluation and long-term aqueous storage of released N-linked glycan stocks Support Protocol: Preparation of single-use glycan aliquots and pre-injection quality control.