Deuterated Cross-Link Identification

Potential cross-links were identified by using the MassMatrix Database Search Engine [52 (link)] using the following search parameters: maximum of two missed tryptic cleavage sites, carbamidomethylation of cysteine and oxidation of methionine residues as variable modifications, mass accuracy filter of 10 p.p.m. for precursor ions and 0.8 Da for fragment ions, minimum pp and pp₂ values of 5.0, minimum pp_tag of 1.3 and maximum number of cross-links per peptide=1.
Two database searches were performed for (i) deuterated and (ii) non-deuterated cross-linked peptides, respectively. Potential cross-links were validated by (i) the presence of the d4/d0-BS3 generated peak pair in the MS spectra and (ii) by the quality of the MSMS spectrum assessed manually.
Extracted ion chromatograms were generated separately for light (d0) and heavy (d4) peaks of the peak pair using XCalibur software v2.1 (Thermo Scientific). Cross-linking ratios were calculated from the peak area of the extracted ion chromatograms.

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Beilsten-Edmands V., Gordiyenko Y., Kung J.C., Mohammed S., Schmidt C, & Robinson C.V. (2015). eIF2 interactions with initiator tRNA and eIF2B are regulated by post-translational modifications and conformational dynamics. Cell Discovery, 1, 15020-.

Publication 2015

XCalibur software v2.1

Cleavage Cysteine Light Methionine Peptides Tryptic

Corresponding Organization :

Other organizations : University of Oxford

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Variable analysis

independent variables

Deuteration
Tryptic cleavage sites (maximum of two missed)
Carbamidomethylation of cysteine residues
Oxidation of methionine residues

dependent variables

Presence of the d4/d0-BS3 generated peak pair in the MS spectra
Quality of the MSMS spectrum assessed manually
Cross-linking ratios calculated from the peak area of the extracted ion chromatograms

control variables

Mass accuracy filter of 10 p.p.m. for precursor ions
Mass accuracy filter of 0.8 Da for fragment ions
Minimum pp and pp2 values of 5.0
Minimum pptag of 1.3
Maximum number of cross-links per peptide = 1

controls

Two database searches were performed for (i) deuterated and (ii) non-deuterated cross-linked peptides, respectively.

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