Abstract
Quantitative cross-linking mass spectrometry (QCLMS) reveals structural detail on altered protein states in solution. On its way to becoming a routine technology, QCLMS could benefit from data-independent acquisition (DIA), which generally enables greater reproducibility than data-dependent acquisition (DDA) and increased throughput over targeted methods. Therefore, here we introduce DIA to QCLMS by extending a widely used DIA software, Spectronaut, to 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 10%, with manual correction of peak selection and boundaries for PSMs in the lower quartile of individual CV values. This compares favorably to DDA where we quantified cross-links across triplicates with a CV of 66%, for a single protein. We found DIA-QCLMS to be capable of detecting changing abundances of cross-linked peptides in complex mixtures, despite the ratio compression encountered when increasing sample complexity through the addition of E. coli cell lysate as matrix. In conclusion, the DIA software Spectronaut can now be used in cross-linking and DIA is indeed able to improve QCLMS.
Highlights
Quantitative cross-linking mass spectrometry (QCLMS) reveals structural detail on altered protein states in solution
On its way to becoming a routine technology, quantitative cross-linking mass spectrometry (QCLMS) could benefit from data-independent acquisition (DIA), which generally enables greater reproducibility than data-dependent acquisition (DDA) and increased throughput over targeted methods
We found DIAQCLMS to be capable of detecting changing abundances of cross-linked peptides in complex mixtures, despite the ratio compression encountered when increasing sample complexity through the addition of E. coli cell lysate as matrix
Summary
We present here a simple, user friendly and automated new quantitative cross-linking mass spectrometry (QCLMS) workflow comprising data-independent acquisition (DIA) for acquiring mass spectrometry data and Spectronaut, one of the leading DIA analysis tools. Data-dependent acquisition (DDA) results in poor reproducibility for low abundance proteins or peptides (38 – 40) and is not ideal for the typically low abundance cross-linked peptides Targeted proteomic strategies such as SRM (MRM) or PRM excel for less abundant peptides [41,42,43,44,45]. Data-independent acquisition (DIA) promises a solution to all these challenges by requiring minimal assay development and allowing large scale quantitative analysis with high reproducibility [48, 49]. This has not yet been exploited in QCLMS because of current software restrictions regarding crosslinked peptides. We determined the accuracy and reproducibility of our DIAQCLMS workflow at both MS1 as well as MS2 level, using a mix of seven proteins, each cross-linked using bis[sulfosuccinimidyl] suberate (BS3), and E. coli cell lysate as matrix
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