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Taxonomic group: fungi / Ascomycota (Phylum: Ascomycota)

NCBI PubMed ID: 32252907
Publication DOI: 10.1016/j.aca.2020.02.042
Journal NLM ID: 0370534
Correspondence: a.gargano

uva.nl
Institutions: Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands, Centre for Analytical Sciences Amsterdam, Amsterdam, The Netherlands, Division of BioAnalytical Chemistry, Amsterdam Institute for Molecular and Life Sciences, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands, van‘t Hoff Institute for Molecular Science, University of Amsterdam, Amsterdam, The Netherlands, DSM Biotechnology Center, Analysis Department, Delft, The Netherlands

Many industrial enzymes exhibit macro- and micro-heterogeneity due to co-occurring post-translational modifications. The resulting proteoforms may have different activity and stability and, therefore, the characterization of their distributions is of interest in the development and monitoring of enzyme products. Protein glycosylation may play a critical role as it can influence the expression, physical and biochemical properties of an enzyme. We report the use of hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) to profile intact glycoform distributions of high mannose-type N-glycosylated proteins, using an industrially produced fungal lipase for the food industry as an example. We compared these results with conventional reversed phase LC-MS (RPLC-MS) and sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE). HILIC appeared superior in resolving lipase heterogeneity, facilitating mass assignment of N-glycoforms and sequence variants. In order to understand the glycoform selectivity provided by HILIC, fractions from the four main HILIC elution bands for lipase were taken and subjected to SDS-PAGE and bottom-up proteomic analysis. These analyses enabled the identification of the most abundant glycosylation sites present in each fraction and corroborated the capacity of HILIC to separate protein glycoforms based on the number of glycosylation sites occupied. Compared to RPLC-MS, HILIC-MS reducted the sample complexity delivered to the mass spectrometer, facilitating the assignment of the masses of glycoforms and sequence variants as well as increasing the number of glycoforms detected (69 more proteoforms, 177% increase). The HILIC-MS method required relatively short analysis time (<30 min), in which over 100 glycoforms were distinguished. We suggest that HILIC(-MS) can be a valuable tool in characterizing bioengineering processes aimed at steering protein glycoform expression as well as to check the consistency of product batches.

high-resolution mass spectrometry, HILIC, high mannose-type N-glycosylation, intact protein analysis

Structure type: oligomer
Location inside paper: p. 75, Fig. 5
Compound class: polysaccharide, mannan
Contained glycoepitopes: IEDB_115576,IEDB_123886,IEDB_130701,IEDB_135813,IEDB_136104,IEDB_137340,IEDB_137485,IEDB_140116,IEDB_141111,IEDB_141793,IEDB_141807,IEDB_141828,IEDB_141829,IEDB_141830,IEDB_141831,IEDB_143632,IEDB_144983,IEDB_151079,IEDB_151531,IEDB_152206,IEDB_153212,IEDB_153220,IEDB_153762,IEDB_153763,IEDB_164174,IEDB_164176,IEDB_187201,IEDB_187238,IEDB_429156,IEDB_540671,IEDB_548907,IEDB_857732,IEDB_857734,IEDB_857735,IEDB_983930,SB_136,SB_191,SB_196,SB_197,SB_198,SB_33,SB_44,SB_53,SB_67,SB_72,SB_73,SB_74,SB_77,SB_85

Methods: SDS-PAGE, acid hydrolysis, HILIC-MS, precipitation, centrifugation, gel electrophoresis, RP-LC-MS

NCBI Taxonomy refs (TaxIDs): 5061
Reference(s) to other database(s): GTC:G65463VQ
Show glycosyltransferases

There are 64 chemically distinct structures. Please, select:

CC(=O)N[C@H]1[C@H](O[C@@H]2[C@@H](CO)O[C@@H](O)[C@H](NC(C)=O)[C@H]2O)O[C@H](CO)[C@@H](O[C@@H]2O[C@H](CO[C@H]3O[C@H](CO[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O)[C@@H](O)[C@H](O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O)[C@@H]3O)[C@@H](O)[C@H](O[C@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]3O[C@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]3O[C@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]3O[C@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]3O[C@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]3O)[C@@H]2O)[C@@H]1O

SVG MW SMILES 3D mol Ionize

Warning = sub-repeat ending at aDManp: unknown unit count was assumed to be 3