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Measuring the hydrodynamic radii of peptides and proteins with an unmodified LC-ESI-MS instrument operating in a Taylor dispersion regime

ABSTRACT: Native ion mobility – mass spectrometry has been well established in recent years as a structural biology method. However, it is still useful to combine gas-phase techniques with complementary solution-phase measurements, for example hydrodynamic radius (Rh) determination. A common method to measure protein Rh is the use of Taylor dispersion analysis (TDA), which relies on measuring the longitudinal diffusion of an analyte in a laminar flow within a capillary. This type of analysis most often relies on spectroscopic (e.g., fluorescence-based) detection, although a few studies have reported the use of custom-built or modified fluidics systems coupled with MS-based detection. Here, we have repurposed a standard, unmodified LC-MS system, operated without a column, to measure hydrodynamic radii of peptides and proteins, including noncovalent complexes, ranging from 300 Da to 133 kDa. The Rh values we measured showed excellent correlation with gas-phase collision cross-sections as well as Rh values measured with a conventional TDA instrument with fluorescence-based detection. Notably, due to the mass-selective readout of our method, which is based on analysis of extracted ion chromatograms, we were able to perform multiplexed Rh measurements of several proteins in a single experiment, and also measured apparent Rh values for different oligomeric states of BSA and concanavalin A. Consistent with prior literature, the latter data suggested that concanavalin A primarily exists as a dimer and tetramer in solution, with monomer signals being largely due to dissociation during electrospray ionisation. We have named our method Hydrodynamic Radii from an Unmodified Liquid Chromatography - Mass Spectrometer (HYDRAULIC-MS).

INSTRUMENT(S): Synapt MS

ORGANISM(S): Bos Taurus (bovine) Homo Sapiens (human) Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Testis

SUBMITTER: Jonathan Eisert

LAB HEAD: Prof. Frederik Lermyte

PROVIDER: PXD062208 | Pride | 2025-07-10

REPOSITORIES: Pride

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Project description:Protein glycolysation is an essential posttranslational modification in eukaryotic cells. In pathogenic yeasts, it is involved in a large number of biological processes, such as protein folding quality control, cell viability and host/pathogen relationships. A link between protein glycosylation and apoptosis was established by the analysis of the phenotypes of oligosaccharyltransferase mutants in budding yeast. However, little is known about the contribution of glycosylation modifications to the adaptive response to apoptosis inducers. The cysteine protease metacaspase Mca1p plays a key role in the apoptotic response in Candida albicans triggered by the quorum sensing molecule farnesol. We subjected wild-type and mca1-deletion strains to farnesol stress and then studied the early phase of apoptosis release in quantitative glycoproteomics and glycomics experiments on cell-free extracts essentially devoid of cell walls. We identified and characterized 62 new glycosylated peptides with their glycan composition: 17 N-glycosylated, 45 O-glycosylated, and 81 additional sites of N-glycosylation. The glycosylated proteins were predicted as located mostly in the “membrane” and “cell wall” compartments, but they were also related to the “nucleus” and the “mitochondrial” compartments. They were found to be involved in the control of protein folding, cell wall integrity and cell cycle regulation. We showed a general increase in the O-glycosylation of proteins in the mca1 deletion strain after farnesol challenge. We identified 44 new putative protein substrates of the metacaspase in the glycoprotein fraction enriched on concanavalin A. Most of these substrates are involved in protein folding or protein resolubilization and in mitochondrial functions. We show here that key Mca1p substrates, such as Cdc48p or Ssb1p, involved in degrading misfolded glycoproteins and in the protein quality control system, are themselves differentially glycosylated. We found putative substrates, such as Bgl2p, Srb1p or Ugp1p, that are involved in the biogenesis of glycans. We validated as a substrate of Mca1p the endo-beta-1,3-glucanase Bgl2p which is involved in the incorporation of newly synthetized mannoproteins in the cell wall. Our findings highlight a new role of the metacaspase in amplifying cell death processes by affecting several critical protein quality control systems through the alteration of the protein glycosylation machinery.

			Action	DRS
	HYDRAULIC-MS.zip	Other
	IM-MS.zip	Other
	checksum.txt	Txt
	excel.xlsx	Xlsx

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Measuring the hydrodynamic radii of peptides and proteins with an unmodified LC-ESI-MS instrument operating in a Taylor dispersion regime

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