Data-independent acquisition improves quantitative cross-linking mass spectrometry

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

Light-Induced Orthogonal Fragmentation of Crosslinked Peptides

Crosslinking mass spectrometry provides pivotal information on the structure and interaction of proteins. MS-cleavable crosslinkers are regarded as a cornerstone for the analysis of complex mixtures. Yet they fragment under similar conditions as peptides, leading to mixed fragmentation spectra of the crosslinker and peptide. This hampers selecting individual peptides for their independent identification. Here, we introduce orthogonal cleavage using ultraviolet photodissociation (UVPD) to increase crosslinker over peptide fragmentation. We designed and synthesized a crosslinker that can be cleaved at 213 nm in a commercial mass spectrometer configuration. In an analysis of crosslinked Escherichia coli lysate, the crosslinker-to-peptide fragment intensity ratio increases from nearly 1 for a conventionally cleavable crosslinker to 5 for the UVPD-cleavable crosslinker. This largely increased the sensitivity of selecting the individual peptides for MS3, even more so with an improved doublet detection algorithm. Data are available via ProteomeXchange with identifier PXD040267.<br/

Light-Induced Orthogonal Fragmentation of Crosslinked Peptides

Crosslinking mass spectrometry provides pivotal information on the structure and interaction of proteins. MS-cleavable crosslinkers are regarded as a cornerstone for the analysis of complex mixtures. Yet they fragment under similar conditions as peptides, leading to mixed fragmentation spectra of the crosslinker and peptide. This hampers selecting individual peptides for their independent identification. Here, we introduce orthogonal cleavage using ultraviolet photodissociation (UVPD) to increase crosslinker over peptide fragmentation. We designed and synthesized a crosslinker that can be cleaved at 213 nm in a commercial mass spectrometer configuration. In an analysis of crosslinked Escherichia coli lysate, the crosslinker-to-peptide fragment intensity ratio increases from nearly 1 for a conventionally cleavable crosslinker to 5 for the UVPD-cleavable crosslinker. This largely increased the sensitivity of selecting the individual peptides for MS3, even more so with an improved doublet detection algorithm. Data are available via ProteomeXchange with identifier PXD040267.<br/

Optimizing the Parameters Governing the Fragmentation of Cross-Linked Peptides in a Tribrid Mass Spectrometer

We compared the five different ways of fragmentation available on a tribrid mass spectrometer and optimized their collision energies with regard to optimal sequence coverage of cross-linked peptides. We created a library of bis­(sulfosuccinimidyl)­suberate (BS3/DSS) cross-linked precursors, derived from the tryptic digests of three model proteins (Human Serum Albumin, creatine kinase, and myoglobin). This enabled in-depth targeted analysis of the fragmentation behavior of 1065 cross-linked precursors using the five fragmentation techniques: collision-induced dissociation (CID), beam-type CID (HCD), electron-transfer dissociation (ETD), and the combinations ETciD and EThcD. EThcD gave the best sequence coverage for cross-linked <i>m</i>/<i>z</i> species with high charge density, while HCD was optimal for all others. We tested the resulting data-dependent decision tree against collision energy-optimized single methods on two samples of differing complexity (a mix of eight proteins and a highly complex ribosomal cellular fraction). For the high complexity sample the decision tree gave the highest number of identified cross-linked peptide pairs passing a 5% false discovery rate (on average ∼21% more than the second best, HCD). For the medium complexity sample, the higher speed of HCD proved decisive. Currently, acquisition speed plays an important role in allowing the detection of cross-linked peptides against the background of linear peptides. Enrichment of cross-linked peptides will reduce this role and favor methods that provide spectra of higher quality. Data are available via ProteomeXchange with identifier PXD006131

xiSPEC_ms_parser

Back-end parser for xiSPEC mass spectrometry visualization tool. xiSPEC is an open source collaborative initiative available in the GitHub repositories (front-end: https://github.com/ Rappsilber-Laboratory/xiSPEC ; back-end: https://github.com/Rappsilber-Laboratory/xiSPEC_ms_parser ). xiAnnotator is an open source collaborative initiative available in the GitHub repository (https://github.com/RappsilberLaboratory/xiAnnotator ). All of which are freely available under the Apache License v2.0.We present xiSPEC, a standard compliant, next-generation web-based spectrum viewer for visualizing, analyzing and sharing mass spectrometry data.Kolbowski L, Combe C, 2018, xiSPEC_ms_parser, Github, https://github.com/Rappsilber-Laboratory/xiSPEC_ms_parse

Source Code for xiSPEC open source

xiSPEC is an open source collaborative initiative available in the GitHub repositories (front-end: https://github.com/ Rappsilber-Laboratory/xiSPEC ; back-end: https://github.com/Rappsilber-Laboratory/xiSPEC_ms_parser ). xiAnnotator is an open source collaborative initiative available in the GitHub repository (https://github.com/RappsilberLaboratory/xiAnnotator ). All of which are freely available under the Apache License v2.0.We present xiSPEC, a standard compliant, next-generation web-based spectrum viewer for visualizing, analyzing and sharing mass spectrometry data.Kolbowski L, Combe C, 2018, Source Code for xiSPEC, Github, https://github.com/Rappsilber-Laboratory/xiSPE

Cleavable Cross-Linkers Redefined by a Novel MS³‑Trigger Algorithm

Cross-linking mass spectrometry (MS) is currently transitioning from a routine tool in structural biology to enabling structural systems biology. MS-cleavable cross-linkers could substantially reduce the associated search space expansion by allowing a MS3-based approach for identifying cross-linked peptides. However, MS2 (MS/MS)-based approaches currently outperform approaches utilizing MS3. We show here that the sensitivity and specificity of triggering MS3 have been hampered algorithmically. Our four-step MS3-trigger algorithm greatly outperformed currently employed methods and comes close to reaching the theoretical limit

xiAnnotator

Java application backend service for the annotation of spectra data. xiAnnotator is an open source collaborative initiative available in the GitHub repository. xiSPEC is an open source collaborative initiative available in the GitHub repositories (front-end: https://github.com/ Rappsilber-Laboratory/xiSPEC ; back-end: https://github.com/Rappsilber-Laboratory/xiSPEC_ms_parser ). xiAnnotator is an open source collaborative initiative available in the GitHub repository (https://github.com/RappsilberLaboratory/xiAnnotator ). All of which are freely available under the Apache License v2.0.We present xiSPEC, a standard compliant, next-generation web-based spectrum viewer for visualizing, analyzing and sharing mass spectrometry data.Kolbowski L, Combe C, 2018, xiAnnotator, Github, https://github.com/Rappsilber-Laboratory/xiAnnotato