Reanalysis of a synthetic cross-linked peptide library with MaxLynx
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Streptococcus Pyogenes Abc020006030
SUBMITTER: 
Sule Yilmaz
LAB HEAD: Juergen Cox
PROVIDER: PXD027159 | Pride | 2022-01-11
REPOSITORIES: Pride
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Project description:Reanalysis of a synthetic crosslinked peptide library dataset. The three replicates of the XL-MS experiment with the non-cleavable cross-linker DSS from the dataset published in Beveridge et al., Nature Commun., 2020 (PRIDE project PXD014337) were searched with the XL-MS identification tool OpenPepXL for benchmarking purposes.
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Project description:The application of non-cleavable cross-linkers to complex samples in XL-MS workflows is limited by the n² problem, a quadratic expansion of the search space with increasing database size. Here, a peptide-focused approach is proposed, for which cross-linking peptide candidates are identified in a parallel experiment by using a thiol-cleavable cross-linker with equal reactivity. Samples are reduced and alkylated, thereby releasing cross-linked peptides with a variable modification on the initially cross-linked residue. Modified peptides are identified by database search and concatenated to a peptide database, which is finally used for the analysis of the sample cross-linked with a non-cleavable cross-linker. This way, the search space is dramatically reduced leading to a higher sensitivity, because there is less chance for random hits to false positive sequences. The approach was benchmarked on cross-linked purified protein complexes (20 S Proteasome, yeast PolII, and TFIIH), and in vivo cross-linked bacteria (Bacillus subtilis, Bacillus cereus) by comparing it to the conventional approach of searching against the protein sequences.
2020-03-26 | PXD016554 | Pride
Project description:The endogenous cellular prion protein (PrPC) can misfold into the scrapie isoform (PrPSc) and cause fatal infectious diseases. Despite significant research on the prion protein, both its normal function and whether alterations to that function play a critical role in prion diseases remain unknown. The protein consists of a predominantly alpha-helical C-terminal domain and an unstructured N-terminal domain that can coordinate Cu2+. Previous studies using NMR and EPR have revealed a tertiary association between the N-terminal domain and the C-terminal domain that we have hypothesized to be critical to the protein’s normal function. Here we investigated and quantified the inter-domain interactions within three different prion variants (wild type recombinant mouse PrPC, mutant delta central region (ΔCR), and disease mutant (E199K) after chemical cross-linking with a newly designed MS-cleavable reagent 1-(4-((2,5-Dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-4-(2-(3-methyl-3H-diazirin-3-yl)ethyl)-1,4-diazabicyclo[2.2.2] octane-1,4-diium (APDC4), followed by nHPLC(RP) and tandem MS analysis.
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Project description:Numerous reagents have been developed to enable chemical proteomic analysis of small molecule-protein interactomes. However, the performance of these reagents has not been systematically evaluated and compared. Herein, we report our efforts to conduct a parallel assessment of two widely-used chemically-cleavable linkers equipped with dialkoxydiphenylsilane (DADPS linker) and azobenzene (AZO linker) moieties. Profiling a cellular cysteinome using iodoacetamide alkyne probe demonstrated a significant discrepancy between the experimental results obtained through the application of each of the reagents. To better understand the source of observed discrepancy, a mass tolerant database search strategy using MSFragger software was performed. This resulted in identifying a previously unreported artifactual modification on the residual mass of the azobenzene linker. Furthermore, we conducted a comparative analysis of enrichment modes using both cleavable linkers. This effort determined that enrichment of proteolytic digests yielded a far greater number of identified cysteine residues than the enrichment conducted prior to protein digest. Inspired by recent studies where multiplexed quantitative labeling strategies were applied to cleavable biotin linkers, we combined this further optimized protocol using the DADPS cleavable linker with tandem mass tag (TMT) labeling to profile the FDA-approved covalent EGFR kinase inhibitor dacomitinib against the cysteinome of an epidermoid cancer cell line. Our analysis resulted in the detection and quantification of over 10,000 unique cysteine residues, a nearly 3-fold increase over previous studies that used cleavable biotin linkers for enrichment. Critically, cysteine residues corresponding to proteins directly as well as indirectly modulated by dacomitinib treatment were identified. Overall, our study suggests that the dialkoxydiphenylsilane linker could be broadly applied wherever chemically cleavable linkers are required for chemical proteomic characterization of cellular proteomes.
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Project description:We have created a synthetic crosslinked peptide library to benchmark crosslinking mass spectrometry search engines. The unique benefit of the library is knowing which identified crosslinks are true and which are false. The data collected from mass spectrometry measurements of the peptide library were used to assess the most frequently used search algorithms. The datasets included will provide an important resource for the crosslinking community to evaluate and optimise search engines, results from which have far-reaching implications.
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Project description:Disuccinimidyl dibutyric urea (DSBU) is a mass spectrometry (MS)-cleavable cross-linker that has been applied to multiple applications in structural biology, ranging from isolated protein complexes to comprehensive system-wide interactomics. DSBU facilitates a rapid and reliable identification of cross-links through the dissociation of its urea group in the gas-phase. In this study, we further advance the structural capabilities of DSBU by twisting the urea group into an imide, thus introducing a novel class of cross-linkers. This modification preserves the MS-cleavability of the amide bond, granted by the two acyl groups of the imide function. The central nitrogen atom enables the introduction of affinity purification tags. Here, we introduce disuccinimidyl dibutyric imide (DSBI) as prototype of this class of cross-linkers. It features a phosphonate handle for immobilized metal ion affinity chromatography (IMAC) enrichment. We detail DSBI synthesis and describe its behavior in solution and in the gas-phase while cross-linking isolated proteins and human cell lysates. DSBI and DSBU cross-links are compared at the same enrichment depths to bridge these two cross-linker classes. We validate DSBI cross-links by mapping them in high-resolution structures of large protein assemblies . The cross-links observed yield insights into the morphology of intrinsically disordered proteins (IDPs) and their complexes. The DSBI linker might spearhead a novel class of MS-cleavable and enrichable cross-linkers
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