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Evaluation of top-down mass spectrometry and ion-mobility spectroscopy as a means of mapping protein-binding motifs within heparin chains.

ABSTRACT: Identifying structural elements within heparin (as well as other glycosaminoglycan) chains that enable their interaction with a specific client protein remains a challenging task due to the high degree of both intra- and inter-chain heterogeneity exhibited by this polysaccharide. The new experimental approach explored in this work is based on the assumption that the heparin chain segments bound to the protein surface will be less prone to collision-induced dissociation (CID) in the gas phase compared to the chain regions that are not involved in binding. Facile removal of the unbound chain segments from the protein/heparin complex should allow the length and the number of sulfate groups within the protein-binding segment of the heparin chain to be determined by measuring the mass of the truncated heparin chain that remains bound to the protein. Conformational integrity of the heparin-binding interface on the protein surface in the course of CID is ensured by monitoring the evolution of collisional cross-section (CCS) of the protein/heparin complexes as a function of collisional energy. A dramatic increase in CCS signals the occurrence of large-scale conformational changes within the protein and identifies the energy threshold, beyond which relevant information on the protein-binding segments of heparin chains is unlikely to be obtained. Testing this approach using a 1 : 1 complex formed by a recombinant form of an acidic fibroblast growth factor (FGF-1) and a synthetic pentasaccharide GlcNS,6S-GlcA-GlcNS,3S,6S-IdoA2S-GlcNS,6S-Me as a model system indicated that a tri-saccharide fragment is the minimal-length FGF-binding segment. Extension of this approach to a decameric heparin chain (dp10) allowed meaningful binding data to be obtained for a 1 : 1 protein/dp10 complex, while the ions representing the higher stoichiometry complex (2 : 1) underwent dissociation via asymmetric charge partitioning without generating truncated heparin chains that remain bound to the protein.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC7160044 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Fecal volatile organic compounds (VOCs) are increasingly considered to be potential noninvasive, diagnostic biomarkers for various gastrointestinal diseases. Knowledge of the influence of sampling conditions on VOC outcomes is limited. We aimed to evaluate the effects of sampling conditions on fecal VOC profiles and to assess under which conditions an optimal diagnostic accuracy in the discrimination between pediatric inflammatory bowel disease (IBD) and controls could be obtained. Fecal samples from de novo treatment-naïve pediatric IBD patients and healthy controls (HC) were used to assess the effects of sampling conditions compared to the standard operating procedure (reference standard), defined as 500 mg of sample mass diluted with 10 mL tap water, using field asymmetric ion mobility spectrometry (FAIMS). A total of 17 IBD (15 CD (Crohn's disease) and 2 UC (ulcerative colitis)) and 25 HC were included. IBD and HC could be discriminated with high accuracy (accuracy = 0.93, AUC = 0.99, p < 0.0001). A smaller fecal sample mass resulted in a decreased diagnostic accuracy (300 mg accuracy = 0.77, AUC = 0.69, p = 0.02; 100 mg accuracy = 0.70, AUC = 0.74, p = 0.003). A loss of diagnostic accuracy was seen toward increased numbers of thaw-freeze cycles (one cycle, accuracy = 0.61, AUC = 0.80, p = 0.0004; two cycles, accuracy = 0.64, AUC = 0.56, p = 0.753; and three cycles, accuracy = 0.57, AUC = 0.50, p = 0.5101) and when samples were kept at room temperature for 180 min prior to analysis (accuracy = 0.60, AUC = 0.51, p = 0.46). Diagnostic accuracy of VOC profiles was not significantly influenced by storage duration differences of 20 months. The application of a 500 mg sample mass analyzed after one thaw-freeze cycle showed the best discriminative accuracy for the differentiation of IBD and HC. VOC profiles and diagnostic accuracy were significantly affected by sampling conditions, underlining the need for the implementation of standardized protocols in fecal VOC analysis.

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Evaluation of top-down mass spectrometry and ion-mobility spectroscopy as a means of mapping protein-binding motifs within heparin chains.

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