Dataset Information

Crosslinking mass spectrometry of the α/β subunit interface in urinary and recombinant human chorionic gonadotropin (hCG)

ABSTRACT: Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone whose urinary (u-hCG) and recombinant (r-hCG) forms differ in glycosylation and stability. To test whether this reflects a structural reorganization of the α/β subunit interface, the two forms were compared by chemical crosslinking mass spectrometry (XL-MS). Crosslinking was performed on the intact, natively glycosylated heterodimers using two complementary chemistries: the lysine-reactive reagent DSS and the zero-length coupling reagent DMTMM (targeting Lys-Asp/Glu pairs, each in biological triplicate for both forms. Identified inter-subunit crosslinks were mapped onto the hCG structure to evaluate whether the assembled core interface is conserved between u-hCG and r-hCG. The dataset comprises the raw LC-MS/MS files and the corresponding crosslink identifications for all conditions and replicates, and accompanies a broader native-MS, ion-mobility and collision-induced-unfolding study of the two hCG forms.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Dinko Soic

LAB HEAD: Alexander Leitner

PROVIDER: PXD080682 | Pride | 2026-07-15

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	260607_LU02_disoic_hCG_01_1.raw	Raw
	260607_LU02_disoic_hCG_01_2.raw	Raw
	260607_LU02_disoic_hCG_01_3.raw	Raw
	260607_LU02_disoic_hCG_02_1.raw	Raw
	260607_LU02_disoic_hCG_02_2.raw	Raw

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Project description:[NiFe]-hydrogenases catalyse the reversible splitting of hydrogen. The catalytic metal centre (NiFe(CN)2CO) is unique in biology, and assembled by an intricate protein machinery, in a process that is still being explored. We hypothesised a structural, ATP-dependent, mechanistic explanation for the assembly of the Fe(CN)2CO fragment via the HypCD complex. We carried out a crosslinking mass spectrometry (crosslinking MS) analysis to study the structure of the HypCD complex, both with (holoprotein) and without (apoprotein) the metal cofactor, and both with and without the addition of ATP. We used the UV-photoactivatable crosslinking reagent sulfo-SDA, which has been shown to have excellent performance when studying dynamic and flexible protein complexes. For photoactivation we used a high-powered LED which enabled the use of exceptionally short reaction times (20 seconds) and gave strikingly clear results. From the resulting crosslinked residue pair patterns identified, we were able to unambiguously distinguish between holoprotein with and without ATP. Crosslinks found in holoprotein, in the absence of ATP, suggested a “closed” protein conformation. When the HypCD holoprotein was crosslinked in the presence of ATP, two distinct crosslink bands were almost entirely absent, indicating that the protein conformation had shifted to an “open” conformation. Interestingly, no shift in protein conformation was evident in the crosslinked apoprotein, which implied that the cofactor was central to protein conformational dynamics. Crosslinking MS data helped to explain, and was in agreement with, protein structures predicted by AlphaFold2 (which were subsequently refined by density functional theory (DFT) modeling for placing the cofactor). Considering all the experimental data from this study led to the conclusion that the binding of ATP alone, not its hydrolysis, is required for the transfer of the Fe(CN)2CO fragment to the apo-hydrogenase large subunit.

Dataset Information

Crosslinking mass spectrometry of the α/β subunit interface in urinary and recombinant human chorionic gonadotropin (hCG)

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