Lutz Fischer

Lutz Fischer
The University of Edinburgh | UoE · Wellcome Trust Centre for Cell Biology

About

78
Publications
6,774
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1,787
Citations
Citations since 2016
58 Research Items
1359 Citations
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2016201720182019202020212022050100150200250
2016201720182019202020212022050100150200250
2016201720182019202020212022050100150200250

Publications

Publications (78)
Preprint
Full-text available
Accurately modeling the structures of proteins and their complexes using artificial intelligence is currently revolutionizing molecular biology. Experimental data enable a candidate-based approach to systematically model novel protein assemblies. Here, we use a combination of in-cell crosslinking mass spectrometry, co-fractionation mass spectrometr...
Article
Full-text available
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 ther...
Preprint
Full-text available
Proteome-wide crosslinking mass spectrometry studies have coincided with the advent of MS-cleavable crosslinkers that can reveal the individual masses of the two crosslinked peptides. However, recently such studies have also been published with non-cleavable crosslinkers suggesting that MS-cleavability is not essential. We therefore examined in det...
Article
Full-text available
Protein-protein interactions govern most cellular pathways and processes, and multiple technologies have emerged to systematically map them. Assessing the error of interaction networks has been a challenge. Crosslinking mass spectrometry is currently widening its scope from structural analyses of purified multi-protein complexes towards systems-wid...
Article
Cross-linking mass spectrometry (MS) has substantially matured as a method over the past 2 decades through parallel development in multiple labs, demonstrating its applicability to protein structure determination, conformation analysis, and mapping protein interactions in complex mixtures. Cross-linking MS has become a much-appreciated and routinel...
Preprint
Full-text available
Crosslinking mass spectrometry (Crosslinking MS) has substantially matured as a method over the last two decades through parallel development in multiple labs, demonstrating its applicability for protein structure determination, conformation analysis and mapping protein interactions in complex mixtures. Crosslinking MS has become a much-appreciated...
Preprint
Full-text available
Crosslinking mass spectrometry is widening its scope from structural analyzes of purified multi-protein complexes towards systems-wide analyzes of protein-protein interactions. Assessing the error in these large datasets is currently a challenge. Using a controlled large-scale analysis of Escherichia coli cell lysate, we demonstrate a reliable fals...
Article
Full-text available
The field of structural biology is increasingly focusing on studying proteins in situ, i.e. in their greater biological context. Crosslinking mass spectrometry is contributing to this effort, typically through the use of MS-cleavable crosslinkers. Here, we apply the popular non-cleavable crosslinker disuccinimidyl suberate to human mitochondria and...
Article
Full-text available
Abstract 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 crossl...
Article
Full-text available
The RNA binding proteome (RBPome) was previously investigated using UV crosslinking and purification of poly(A)‐associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA‐associated protein purification (TRAPP) based on 254 nm UV crosslinking and purification of all RNA–protein complexes using s...
Article
Crosslinking 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 artefacts. Here, we describe the observation of non-covalently-ass...
Preprint
Full-text available
Crosslinking 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 artefacts. Here, we describe the observation of non-covalently-ass...
Preprint
Full-text available
The identification of ever-increasing numbers of RNA species has underlined the importance of robust characterization of bona fide sites of protein-RNA interaction. UV crosslinking can be used to identify precise RNA targets for individual proteins, transcriptome-wide. Here we sought to generate reciprocal data, identifying precise sites of RNA-bin...
Article
Full-text available
Cross-linking/mass spectrometry has undergone a maturation process akin to standard proteomics by adapting key methods such as false discovery rate control and quantification. A poorly evaluated search setting in proteomics is the consideration of multiple (lighter) alternative values for the monoisotopic precursor mass to compensate for possible m...
Preprint
Full-text available
UV crosslinking can be used to identify precise RNA targets for individual proteins, transcriptome-wide. We sought to develop a technique to generate reciprocal data, identifying precise sites of RNA-binding proteome-wide. The resulting technique, total RNA-associated protein purification (TRAPP), was applied to yeast (S. cerevisiae) and bacteria (...
Preprint
Full-text available
We present a concise workflow to enhance the mass spectrometric detection of cross-linked peptides by introducing sequential digestion and the cross-link identification software Xi. Sequential digestion enhances peptide detection by shortening long tryptic peptides while avoiding over-digestion. We demonstrate our simple 12-fraction protocol for cr...
Preprint
Full-text available
Cross-linking/mass spectrometry (CLMS) has gone through a maturation process similar to standard proteomics by adapting key methods such as false discovery rate control and quantification. However, methods for preprocessing mass spectrometric data of cross-linked peptides are currently underexplored. Preprocessing only requires computational effort...
Article
Full-text available
False discovery rate (FDR) estimation is a cornerstone of proteomics that has recently been adapted to cross-linking/mass spectrometry. Here we demonstrate that heterobifunctional cross-linkers, while theoretically different from homobifunctional cross-linkers, need not be considered separately in practice. We develop and then evaluate the impact o...
Data
Derivation of formula. (DOCX)
Article
Full-text available
Quantitative cross-linking/mass spectrometry (QCLMS) is an emerging approach to study conformational changes of proteins and multi-subunit complexes. Distinguishing protein conformations requires reproducibly identifying and quantifying cross-linked peptides. Here we analyzed the variation between multiple cross-linking reactions using bis[sulfosuc...
Article
Full-text available
The first stable version of the Proteomics Standards Initiative mzIdentML open data standard (version 1.1) was published in 2012 - capturing the outputs of peptide and protein identification software. In the intervening years, the standard has become well supported in both commercial and open software, as well as a submission and download format fo...
Article
Full-text available
Cross-linking/mass spectrometry is an increasingly popular approach to obtain structural information on proteins and their complexes in solution. However, methods for error assessment are under current development. We note that false-discovery rates can be estimated at different points during data analysis, and are most relevant for residue or prot...
Article
Full-text available
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 structur...
Article
Full-text available
Quantitative cross-linking/mass spectrometry (QCLMS) probes protein structural dynamics in solution by quantitatively comparing the yields of cross-links between different conformational statuses. We have used QCLMS to understand the final maturation step of the proteasome lid and also to elucidate the structure of complement C3(H2O). Here we bench...
Data
List of identified and quantified cross-linked peptides.
Data
Structural details of cross-links that are enriched in either C3 or C3b.
Data
Annotated best-matched MS2 spectrum for quantified cross-linked peptides.
Data
General spectral library format for pinpoint comma separated values.
Data
List of quantified cross-linked residue pairs.
Data
Structural details of C3/C3b-unique cross-links that are within cross-linkable distance in the crystal structures of both C3 and C3b.
Article
Full-text available
The conceptually simple step from cross-linking/mass spectrometry (CLMS) to quantitative cross-linking/mass spectrometry (QCLMS) is compounded by technical challenges. Currently, quantitative proteomics software is tightly integrated with the protein identification workflow. This prevents automatically quantifying other m/z features in a targeted m...
Article
Full-text available
The slow but spontaneous and ubiquitous formation of C3(H2O), the hydrolytic and conformationally rearranged product of C3, initiates antibody-independent activation of the complement system that is a key first line of antimicrobial defense. The structure of C3(H2O) has not been determined. Here we subjected C3(H2O) to quantitative cross-linking/ma...
Preprint
The slow but spontaneous and ubiquitous formation of C3(H 2 O), the hydrolytic and conformationally rearranged product of C3, initiates antibody-independent activation of the complement system that is a key first line of antimicrobial defense. The structure of C3(H 2 O) has not been determined. Here we subjected C3(H 2 O) to quantitative cross-link...
Preprint
The conceptually simple step from cross-linking/mass spectrometry (CLMS) to quantitative cross-linking/mass spectrometry (QCLMS) is compounded by technical challenges. Currently, quantitative proteomics software is tightly integrated with the protein identification workflow. This prevents automatically quantifying other m/z features in a targeted m...
Preprint
We have developed quantitative cross-linking/mass spectrometry (QCLMS) to interrogate conformational rearrangements of proteins in solution. Our workflow was tested using a structurally well-described reference system, the human complement protein C3 and its activated cleavage product C3b. We found that small local conformational changes affect the...
Preprint
Determining the structure of a protein by any method requires varies contributions from experimental and computational sides. In a recent study, high-density cross-linking/mass spectrometry data in combination with ab initio structure prediction by conformational space search determined the structure of human serum albumin (HSA) domains, with an RM...
Article
Full-text available
Cross-linking/mass spectrometry (CLMS) resolves protein-protein interactions or protein folds by help of distance constraints. Cross-linkers with specific properties such as isotope-labelled or CID-cleavable cross-linkers are in frequent use to simplify the identification of cross-linked peptides. Here, we analyzed the mass spectrometric behavior o...
Article
Full-text available
Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low resolution of cross-linking has so far precluded its use in determining structures de novo. Cross-linking resolution has been typically limited by the chemical selectivity of the standard cross-li...
Article
Full-text available
xiNET is a visualization tool for exploring cross-linking/mass spectrometry results. The interactive maps of the cross-link network that it generates are an extended type of node-link diagram. In these maps xiNET displays: (i) residue resolution positional information including linkage sites and linked peptides; (ii) ambiguous results; (iii) additi...
Article
Full-text available
Dynamic proteins and multi-protein complexes govern most biological processes. Cross-linking/mass spectrometry (CLMS) is increasingly successful in providing residue-resolution data on static proteinaceous structures. Here we investigate the technical feasibility of recording dynamic processes using isotope-labelling for quantitation. We cross-link...
Data
This table provides the accession numbers for all domains annotated for proteins in our dataset. The frequency of each domain in chromosomal and nonchromosomal proteins is also given. These data were used to generate classifier VI. Notably, 92 chromosomal and 120 nonchromosomal proteins had no domains annotated and so could not be segregated based...
Data
Details of the proteins identified across the five proteomic experiments. Column “TF” refers to whether the protein is considered true (chromosomal) or false (nonchromosomal) in the training set. Columns “Localization,” “Function,” “Name” and “Complex” are various annotations we have made to the proteins. “Domain Accessions” are the accession numbe...
Article
Full-text available
Despite many decades of study, mitotic chromosome structure and composition remain poorly characterized. Here, we have integrated quantitative proteomics with bioinformatic analysis to generate a series of independent classifiers that describe the approximately 4,000 proteins identified in isolated mitotic chromosomes. Integrating these classifiers...