MassIVE MSV000087019

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Similarities and differences in the structures and proteoform profiles of the Complement proteins C6 and C7

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Description

In this study, we used an integrative structural MS-based approach combining native MS, glycopeptide-centric MS, in-gel cross-linking MS (IGX-MS) to describe structural and glycosylation features of soluble C7. We compare this data with structural and glycosylation data for C6. For native MS analysis, proteins were buffer-exchanged into 150 mM aqueous ammonium acetate (pH 7.5) and analyzed on a modified Q-Exactive Plus Orbitrap mass spectrometer with extended mass range (EMR). For glyco-peptide centric analysis LC-MS and MS/MS analysis, C6 and C7 were digested by trypsin and a combination of Glu-C and trypsin. The resulting peptide mixtures were desalted, dried, and reconstituted in formic acid before MS analysis. All peptides were separated and analyzed using an Agilent 1290 Infinity HPLC system coupled on-line to an Orbitrap Q Exactive HF mass spectrometer. For the cross-linking analysis C7 gel band was cut from a BN-PAGE and cross-linked in-gel. The cross-linked protein were digested in the gel and data was acquired using an Ultimate 3000 system coupled on-line to an Orbitrap Exploris. The raw native MS spectra were deconvoluted to zero-charge spectra by Intact Mass software (Protein Metrics, CA, USA). The spectra were annotated using an in-house developed R script for semi-automated peak annotation, as described previously. The glycopeptide-centric bottom-up raw data were processed by the software Byonic and Skyline. Cross-linking raw data was searched in Proteome discoverer using the XlinkX node. [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Glycosylation ; Cross-linking MS ; Native MS

Contact

Principal Investigators:
(in alphabetical order) 		Albert J.R. Heck, Biomolecular Mass Spectrometry and Proteomics groups, Utrecht University, N/A
Submitting User: M_Lukassen
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