Data independent acquisition cross-linking/mass spectrometry

ABSTRACT: Quantitative cross-linking/mass spectrometry (QCLMS) provides increasing structural detail on altered protein states in solution. Accurate quantitation is a value in itself but may also be central to elucidating small differences between protein states. Hence, QCLMS could benefit from data independent acquisition (DIA) which generally provides higher reproducibility than data dependent acquisition (DDA) and higher throughput than targeted methods. Therefore we here open DIA to QCLMS by extending a widely used DIA software, Spectronaut to now also accommodate cross-link data. A mixture of seven proteins cross-linked with bis[sulfosuccinimidyl] suberate (BS3) was used to evaluate this workflow. Out of the 414 identified unique residue pairs, 292 (70%) were quantifiable across triplicates with a coefficient of variation (CV) of 9.8%, with manual correction of peak selection and boundaries for PSMs in the lower quartile of individual CV values. This compares favourably to DDA where we previously quantified only 63% of the identified cross-links across triplicates with a CV of 14%, for a single protein and complete manual data curation. DIA QCLMS is promising to detect differential abundance of cross-linked peptides in complex mixtures despite the encountered ratio compression when increasing sample complexity through the addition of E. coli cell lysate as matrix. In conclusion, DIA software Spectronaut can now be used in cross-linking and DIA is indeed able to improve QCLMS.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Equus Caballus (horse) Homo Sapiens (human) Bos Taurus (bovine) Gallus Gallus (chicken) Oryctolagus Cuniculus (rabbit)

SUBMITTER: Fränze Müller  

LAB HEAD: Juri Rappsilber

PROVIDER: PXD011036 | Pride | 2019-01-25

REPOSITORIES: Pride