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.

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.

Improved Peptide Backbone Fragmentation Is the Primary Advantage of MS-Cleavable Crosslinkers.

Proteome-wide crosslinking mass spectrometry studies have coincided with the advent of mass spectrometry (MS)-cleavable crosslinkers that can reveal the individual masses of the two crosslinked peptides. However, recently, such studies have also been published with noncleavable crosslinkers, suggesting that MS-cleavability is not essential. We therefore examined in detail the advantages and disadvantages of using the commonly used MS-cleavable crosslinker, disuccinimidyl sulfoxide (DSSO). Indeed, DSSO gave rise to signature peptide fragments with a distinct mass difference (doublet) for nearly all identified crosslinked peptides. Surprisingly, we could show that it was not these peptide masses that proved the main advantage of MS cleavability of the crosslinker, but improved peptide backbone fragmentation which reduces the ambiguity of peptide identifications. This also holds true for another commonly used MS-cleavable crosslinker, DSBU. We show furthermore that the more intricate MS3-based data acquisition approaches lack sensitivity and specificity, causing them to be outperformed by the simpler and faster stepped higher-energy collisional dissociation (HCD) method. This understanding will guide future developments and applications of proteome-wide crosslinking mass spectrometry.

Ultraviolet Photodissociation of Tryptic Peptide Backbones at 213 nm.

We analyzed the backbone fragmentation behavior of tryptic peptides of a four-protein mixture and of E. coli lysate subjected to ultraviolet photodissociation (UVPD) at 213 nm on a commercially available UVPD-equipped tribrid mass spectrometer. We obtained 15 178 unique high-confidence peptide UVPD spectrum matches by recording a reference beam-type collision-induced dissociation (HCD) spectrum of each precursor, ensuring that our investigation includes a broad selection of peptides, including those that fragmented poorly by UVPD. Type a, b, and y ions were most prominent in UVPD spectra, and median sequence coverage ranged from 5.8% (at 5 ms laser excitation time) to 45.0% (at 100 ms). Overall, the sequence fragment intensity remained relatively low (median: 0.4% (5 ms) to 16.8% (100 ms) of total intensity), and the remaining precursor intensity, high. The sequence coverage and sequence fragment intensity ratio correlated with the precursor charge density, suggesting that UVPD at 213 nm may suffer from newly formed fragments sticking together due to noncovalent interactions. The UVPD fragmentation efficiency therefore might benefit from supplemental activation, as was shown for ETD. Aromatic amino acids, most prominently tryptophan, facilitated UVPD. This points to aromatic tags as possible enhancers of UVPD. Data are available via ProteomeXchange with identifier PXD018176 and on spectrumviewer.org/db/UVPD-213nm-trypPep.

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.

xiSPEC: web-based visualization, analysis and sharing of proteomics data.

We present xiSPEC, a standard compliant, next-generation web-based spectrum viewer for visualizing, analyzing and sharing mass spectrometry data. Peptide-spectrum matches from standard proteomics and cross-linking experiments are supported. xiSPEC is to date the only browser-based tool supporting the standardized file formats mzML and mzIdentML defined by the proteomics standards initiative. Users can either upload data directly or select files from the PRIDE data repository as input. xiSPEC allows users to save and share their datasets publicly or password protected for providing access to collaborators or readers and reviewers of manuscripts. The identification table features advanced interaction controls and spectra are presented in three interconnected views: (i) annotated mass spectrum, (ii) peptide sequence fragmentation key and (iii) quality control error plots of matched fragments. Highlighting or selecting data points in any view is represented in all other views. Views are interactive scalable vector graphic elements, which can be exported, e.g. for use in publication. xiSPEC allows for re-annotation of spectra for easy hypothesis testing by modifying input data. xiSPEC is freely accessible at http://spectrumviewer.org and the source code is openly available on https://github.com/Rappsilber-Laboratory/xiSPEC.

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 m/z 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.