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.

Dual-Bioorthogonal Catalysis by a Palladium Peptide Complex.

Artificial metalloenzymes (ArMs) enrich bioorthogonal chemistry with new-to-nature reactions while limiting metal deactivation and toxicity. This enables biomedical applications such as activating therapeutics in situ. However, while combination therapies are becoming widespread anticancer treatments, dual catalysis by ArMs has not yet been shown. We present a heptapeptidic ArM with a novel peptide ligand carrying a methyl salicylate palladium complex. We observed that the peptide scaffold reduces metal toxicity while protecting the metal from deactivation by cellular components. Importantly, the peptide also improves catalysis, suggesting involvement in the catalytic reaction mechanism. Our work shows how a palladium-peptide homogeneous catalyst can simultaneously mediate two types of chemistry to synthesize anticancer drugs in human cells. Methyl salicylate palladium LLEYLKR peptide (2-Pd) succeeded to simultaneously produce paclitaxel by depropargylation, and linifanib by Suzuki-Miyaura cross-coupling in cell culture, thereby achieving combination therapy on non-small-cell lung cancer (NSCLC) A549 cells.

Anatomy of a crosslinker.

Crosslinking mass spectrometry has become a core technology in structural biology and is expanding its reach towards systems biology. Its appeal lies in a rapid workflow, high sensitivity and the ability to provide data on proteins in complex systems, even in whole cells. The technology depends heavily on crosslinking reagents. The anatomy of crosslinkers can be modular, sometimes comprising combinations of functional groups. These groups are defined by concepts including: reaction selectivity to increase information density, enrichability to improve detection, cleavability to enhance the identification process and isotope-labelling for quantification. Here, we argue that both concepts and functional groups need more thorough experimental evaluation, so that we can show exactly how and where they are useful when applied to crosslinkers. Crosslinker design should be driven by data, not only concepts. We focus on two crosslinker concepts with large consequences for the technology, namely reactive group reaction kinetics and enrichment groups.

Megadalton-sized Dityrosine Aggregates of α-Synuclein Retain High Degrees of Structural Disorder and Internal Dynamics.

Heterogeneous aggregates of the human protein α-synuclein (αSyn) are abundantly found in Lewy body inclusions of Parkinson's disease patients. While structural information on classical αSyn amyloid fibrils is available, little is known about the conformational properties of disease-relevant, non-canonical aggregates. Here, we analyze the structural and dynamic properties of megadalton-sized dityrosine adducts of αSyn that form in the presence of reactive oxygen species and cytochrome c, a proapoptotic peroxidase that is released from mitochondria during sustained oxidative stress. In contrast to canonical cross-ß amyloids, these aggregates retain high degrees of internal dynamics, which enables their characterization by solution-state NMR spectroscopy. We find that intermolecular dityrosine crosslinks restrict αSyn motions only locally whereas large segments of concatenated molecules remain flexible and disordered. Indistinguishable aggregates form in crowded in vitro solutions and in complex environments of mammalian cell lysates, where relative amounts of free reactive oxygen species, rather than cytochrome c, are rate limiting. We further establish that dityrosine adducts inhibit classical amyloid formation by maintaining αSyn in its monomeric form and that they are non-cytotoxic despite retaining basic membrane-binding properties. Our results suggest that oxidative αSyn aggregation scavenges cytochrome c's activity into the formation of amorphous, high molecular-weight structures that may contribute to the structural diversity of Lewy body deposits.

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.

Assessment of chemical-crosslink-assisted protein structure modeling in CASP13.

With the advance of experimental procedures obtaining chemical crosslinking information is becoming a fast and routine practice. Information on crosslinks can greatly enhance the accuracy of protein structure modeling. Here, we review the current state of the art in modeling protein structures with the assistance of experimentally determined chemical crosslinks within the framework of the 13th meeting of Critical Assessment of Structure Prediction approaches. This largest-to-date blind assessment reveals benefits of using data assistance in difficult to model protein structure prediction cases. However, in a broader context, it also suggests that with the unprecedented advance in accuracy to predict contacts in recent years, experimental crosslinks will be useful only if their specificity and accuracy further improved and they are better integrated into computational workflows.

An integrated workflow for crosslinking mass spectrometry.

We present a concise workflow to enhance the mass spectrometric detection of crosslinked peptides by introducing sequential digestion and the crosslink identification software xiSEARCH. Sequential digestion enhances peptide detection by selective shortening of long tryptic peptides. We demonstrate our simple 12-fraction protocol for crosslinked multi-protein complexes and cell lysates, quantitative analysis, and high-density crosslinking, without requiring specific crosslinker features. This overall approach reveals dynamic protein-protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.

Noncovalently Associated Peptides Observed during Liquid Chromatography-Mass Spectrometry and Their Effect on Cross-Link Analyses.

Cross-linking mass spectrometry draws structural information from covalently linked peptide pairs. When these links do not match to previous structural models, they may indicate changes in protein conformation. Unfortunately, such links can also be the result of experimental error or artifacts. Here, we describe the observation of noncovalently associated peptides during liquid chromatography-mass spectrometry analysis, which can easily be misidentified as cross-linked. Strikingly, they often mismatch to the protein structure. Noncovalently associated peptides presumably form during ionization and can be distinguished from cross-linked peptides by observing coelution of the corresponding linear peptides in MS1 spectra, as well as the presence of the individual (intact) peptide fragments in MS2 spectra. To suppress noncovalent peptide formations, increasingly disruptive ionization settings can be used, such as in-source fragmentation.

Protein Tertiary Structure by Crosslinking/Mass Spectrometry.

Observing the structures of proteins within the cell and tracking structural changes under different cellular conditions are the ultimate challenges for structural biology. This, however, requires an experimental technique that can generate sufficient data for structure determination and is applicable in the native environment of proteins. Crosslinking/mass spectrometry (CLMS) and protein structure determination have recently advanced to meet these requirements and crosslinking-driven de novo structure determination in native environments is now possible. In this opinion article, we highlight recent successes in the field of CLMS with protein structure modeling and challenges it still holds.

Small angle X-ray scattering and cross-linking for data assisted protein structure prediction in CASP 12 with prospects for improved accuracy.

Experimental data offers empowering constraints for structure prediction. These constraints can be used to filter equivalently scored models or more powerfully within optimization functions toward prediction. In CASP12, Small Angle X-ray Scattering (SAXS) and Cross-Linking Mass Spectrometry (CLMS) data, measured on an exemplary set of novel fold targets, were provided to the CASP community of protein structure predictors. As solution-based techniques, SAXS and CLMS can efficiently measure states of the full-length sequence in its native solution conformation and assembly. However, this experimental data did not substantially improve prediction accuracy judged by fits to crystallographic models. One issue, beyond intrinsic limitations of the algorithms, was a disconnect between crystal structures and solution-based measurements. Our analyses show that many targets had substantial percentages of disordered regions (up to 40%) or were multimeric or both. Thus, solution measurements of flexibility and assembly support variations that may confound prediction algorithms trained on crystallographic data and expecting globular fully-folded monomeric proteins. Here, we consider the CLMS and SAXS data collected, the information in these solution measurements, and the challenges in incorporating them into computational prediction. As improvement opportunities were only partly realized in CASP12, we provide guidance on how data from the full-length biological unit and the solution state can better aid prediction of the folded monomer or subunit. We furthermore describe strategic integrations of solution measurements with computational prediction programs with the aim of substantially improving foundational knowledge and the accuracy of computational algorithms for biologically-relevant structure predictions for proteins in solution.

Complementary Benzophenone Cross-Linking/Mass Spectrometry Photochemistry.

Use of a heterobifunctional photoactivatable cross-linker, sulfo-SDA (diazirine), has yielded high-density data that facilitated structure modeling of individual proteins. We expand the photoactivatable chemistry toolbox here with a second reagent, sulfo-SBP (benzophenone). This further increases the density of photo-cross-linking to a factor of 20× over conventional cross-linking. Importantly, the two different photoactivatable groups display orthogonal directionality, enabling access to different protein regions, unreachable with a single cross-linker.

Repression of RNA polymerase by the archaeo-viral regulator ORF145/RIP.

Little is known about how archaeal viruses perturb the transcription machinery of their hosts. Here we provide the first example of an archaeo-viral transcription factor that directly targets the host RNA polymerase (RNAP) and efficiently represses its activity. ORF145 from the temperate Acidianus two-tailed virus (ATV) forms a high-affinity complex with RNAP by binding inside the DNA-binding channel where it locks the flexible RNAP clamp in one position. This counteracts the formation of transcription pre-initiation complexes in vitro and represses abortive and productive transcription initiation, as well as elongation. Both host and viral promoters are subjected to ORF145 repression. Thus, ORF145 has the properties of a global transcription repressor and its overexpression is toxic for Sulfolobus. On the basis of its properties, we have re-named ORF145 RNAP Inhibitory Protein (RIP).

Optimized Fragmentation Regime for Diazirine Photo-Cross-Linked Peptides.

Cross-linking/mass spectrometry has evolved into a robust technology that reveals structural insights into proteins and protein complexes. We leverage a new tribrid instrument with improved fragmentation capacities in a systematic comparison to identify which fragmentation method would be best for the identification of cross-linked peptides. Specifically, we explored three fragmentation methods and two combinations: collision-induced dissociation (CID), beam-type CID (HCD), electron-transfer dissociation (ETD), ETciD, and EThcD. Trypsin-digested, SDA-cross-linked human serum albumin (HSA) served as a test sample, yielding over all methods and in triplicate analysis in total 2602 matched PSMs and 1390 linked residue pairs at 5% false discovery rate, as confirmed by the crystal structure. HCD wins in number of matched peptide-spectrum-matches (958 PSMs) and identified links (446). CID is most complementary, increasing the number of identified links by 13% (58 links). HCD wins together with EThcD in cross-link site calling precision, with approximately 62% of sites having adjacent backbone cleavages that unambiguously locate the link in both peptides, without assuming any cross-linker preference for amino acids. Overall quality of spectra, as judged by sequence coverage of both peptides, is best for EThcD for the majority of peptides. Sequence coverage might be of particular importance for complex samples, for which we propose a data dependent decision tree, else HCD is the method of choice. The mass spectrometric raw data has been deposited in PRIDE (PXD003737).

Blind Evaluation of Hybrid Protein Structure Analysis Methods based on Cross-Linking.

Hybrid methods combine experimental data and computational modeling to analyze protein structures that are elusive to structure determination. To spur the development of hybrid methods, we propose to test them in the context of the CASP experiment and would like to invite experimental groups to participate in this initiative.

Blind testing of cross-linking/mass spectrometry hybrid methods in CASP11.

Hybrid approaches combine computational methods with experimental data. The information contained in the experimental data can be leveraged to probe the structure of proteins otherwise elusive to computational methods. Compared with computational methods, the structures produced by hybrid methods exhibit some degree of experimental validation. In spite of these advantages, most hybrid methods have not yet been validated in blind tests, hampering their development. Here, we describe the first blind test of a specific cross-link based hybrid method in CASP. This blind test was coordinated by the CASP organizers and utilized a novel, high-density cross-linking/mass-spectrometry (CLMS) approach that is able to collect high-density CLMS data in a matter of days. This experimental protocol was developed in the Rappsilber laboratory. This approach exploits the chemistry of a highly reactive, photoactivatable cross-linker to produce an order of magnitude more cross-links than homobifunctional cross-linkers. The Rappsilber laboratory generated experimental CLMS data based on this protocol, submitted the data to the CASP organizers which then released this data to the CASP11 prediction groups in a separate, CLMS assisted modeling experiment. We did not observe a clear improvement of assisted models, presumably because the properties of the CLMS data-uncertainty in cross-link identification and residue-residue assignment, and uneven distribution over the protein-were largely unknown to the prediction groups and their approaches were not yet tailored to this kind of data. We also suggest modifications to the CLMS-CASP experiment and discuss the importance of rigorous blind testing in the development of hybrid methods. Proteins 2016; 84(Suppl 1):152-163. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Blind testing cross-linking/mass spectrometry under the auspices of the 11th critical assessment of methods of protein structure prediction (CASP11).

Determining the structure of a protein by any method requires various contributions from experimental and computational sides. In a recent study, high-density cross-linking/mass spectrometry (HD-CLMS) data in combination with ab initio structure prediction determined the structure of human serum albumin (HSA) domains, with an RMSD to X-ray structure of up to 2.5 Å, or 3.4 Å in the context of blood serum. This paper reports the blind test on the readiness of this technology through the help of Critical Assessment of protein Structure Prediction (CASP). We identified between 201-381 unique residue pairs at an estimated 5% FDR (at link level albeit with missing site assignment precision evaluation), for four target proteins. HD-CLMS proved reliable once crystal structures were released. However, improvements in structure prediction using cross-link data were slight. We identified two reasons for this. Spread of cross-links along the protein sequence and the tightness of the spatial constraints must be improved. However, for the selected targets even ideal contact data derived from crystal structures did not allow modellers to arrive at the observed structure. Consequently, the progress of HD-CLMS in conjunction with computational modeling methods as a structure determination method, depends on advances on both arms of this hybrid approach.

Serum Albumin Domain Structures in Human Blood Serum by Mass Spectrometry and Computational Biology.

Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low density of cross-link data has so far precluded its use in determining structures de novo. Cross-linking density has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4'-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high density photo-cross-linking data with conformational space search to investigate the structure of human serum albumin domains, from purified samples, and in its native environment, human blood serum. Our approach is able to determine human serum albumin domain structures with good accuracy: root-mean-square deviation to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on human serum albumin demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692.

Influence of the chirality of short peptide supramolecular hydrogels in protein crystallogenesis.

For the first time the influence of the chirality of the gel fibers in protein crystallogenesis has been studied. Enantiomeric hydrogels 1 and 2 were tested with model proteins lysozyme and glucose isomerase and a formamidase extracted from B. cereus. Crystallization behaviour and crystal quality of these proteins in both hydrogels are presented and compared.