RESUMEN
Cross-linking mass spectrometry (XL-MS) is a universal tool for probing structural dynamics and protein-protein interactions in vitro and in vivo. Although cross-linked peptides are naturally less abundant than their unlinked counterparts, recent experimental advances improved cross-link identification by enriching the cross-linker-modified peptides chemically with the use of enrichable cross-linkers. However, mono-links (i.e., peptides modified with a hydrolyzed cross-linker) still hinder efficient cross-link identification since a large proportion of measurement time is spent on their MS2 acquisition. Currently, cross-links and mono-links cannot be separated by sample preparation techniques or chromatography because they are chemically almost identical. Here, we found that based on the intensity ratios of four diagnostic peaks when using PhoX/tBu-PhoX cross-linkers, cross-links and mono-links can be partially distinguished. Harnessing their characteristic intensity ratios for real-time library search (RTLS)-based triggering of high-resolution MS2 scans increased the number of cross-link identifications from both single protein samples and intact E. coli cells. Specifically, RTLS improves cross-link identification from unenriched samples and short gradients, emphasizing its advantages in high-throughput approaches and when instrument time or sample amount is limited.
Asunto(s)
Escherichia coli , Péptidos , Péptidos/química , Proteínas/química , Espectrometría de Masas/métodos , Reactivos de Enlaces Cruzados/químicaRESUMEN
Cross-linking mass spectrometry (XL-MS) is a powerful method for the investigation of protein-protein interactions (PPI) from highly complex samples. XL-MS combined with tandem mass tag (TMT) labeling holds the promise of large-scale PPI quantification. However, a robust and efficient TMT-based XL-MS quantification method has not yet been established due to the lack of a benchmarking dataset and thorough evaluation of various MS parameters. To tackle these limitations, we generate a two-interactome dataset by spiking in TMT-labeled cross-linked Escherichia coli lysate into TMT-labeled cross-linked HEK293T lysate using a defined mixing scheme. Using this benchmarking dataset, we assess the efficacy of cross-link identification and accuracy of cross-link quantification using different MS acquisition strategies. For identification, we compare various MS2- and MS3-based XL-MS methods, and optimize stepped higher energy collisional dissociation (HCD) energies for TMT-labeled cross-links. We observed a need for notably higher fragmentation energies compared to unlabeled cross-links. For quantification, we assess the quantification accuracy and dispersion of MS2-, MS3-, and synchronous precursor selection-MS3-based methods. We show that a stepped HCD-MS2 method with stepped collision energies 36-42-48 provides a vast number of quantifiable cross-links with high quantification accuracy. This widely applicable method paves the way for multiplexed quantitative PPI characterization from complex biological systems.
Asunto(s)
Escherichia coli , Células HEK293 , Humanos , Espectrometría de Masas/métodosRESUMEN
MOTIVATION: We present a new software-tool allowing an easy visualization of fragment ions and thus a rapid evaluation of key experimental parameters on the sequence coverage obtained for the MS/MS (tandem mass spectrometry) analysis of intact proteins. Our tool can process data obtained from various deconvolution and fragment assignment software. RESULTS: We demonstrate that TDFragMapper can rapidly highlight the experimental fragmentation parameters that are critical to the characterization of intact proteins of various size using top-down proteomics. AVAILABILITY AND IMPLEMENTATION: TDFragMapper, a demonstration video and user tutorial are freely available for academic use at https://msbio.pasteur.fr/tdfragmapper; all data are thus available from the ProteomeXchange consortium (identifier PXD024643). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Proteómica , Espectrometría de Masas en Tándem , Proteómica/métodos , Proteínas/química , Programas InformáticosRESUMEN
Pathogen identification is crucial to confirm bacterial infections and guide antimicrobial therapy. Although MALDI-TOF mass spectrometry (MS) serves as foundation for tools that enable rapid microbial identification, some bacteria remain challenging to identify. We recently showed that top-down proteomics (TDP) could be used to discriminate closely related enterobacterial pathogens (Escherichia coli, Shigella, and Salmonella) that are indistinguishable with tools rooted in the MALDI-TOF MS approach. Current TDP diagnostic relies on the identification of specific proteoforms for each species through a database search. However, microbial proteomes are often poorly annotated, which complicates the large-scale identification of proteoforms and leads to many unidentified high-quality mass spectra. Here, we describe a new computational pipeline called DiagnoTop that lists discriminative spectral clusters found in TDP data sets that can be used for microbial diagnostics without database search. Applied to our enterobacterial TDP data sets, DiagnoTop could easily shortlist high-quality discriminative spectral clusters, leading to increased diagnostic power. This pipeline opens new perspectives in clinical microbiology and biomarker discovery using TDP.
Asunto(s)
Bacterias/química , Bacterias/patogenicidad , Biología Computacional/métodos , Programas Informáticos , Espectrometría de Masas en Tándem/métodos , Bases de Datos Factuales , Enterobacteriaceae/química , Enterobacteriaceae/patogenicidad , Bases del Conocimiento , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Flujo de TrabajoRESUMEN
The current technique used for microbial identification in hospitals is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). However, it suffers from important limitations, in particular, for closely related species or when the database used for the identification lacks the appropriate reference. In this work, we set up a liquid chromatography (LC)-MS/MS top-down proteomics platform, which aims at discriminating closely related pathogenic bacteria through the identification of specific proteoforms. Using Escherichia coli as a model, all steps of the workflow were optimized: protein extraction, on-line LC separation, MS method, and data analysis. Using optimized parameters, about 220 proteins, corresponding to more than 500 proteoforms, could be identified in a single run. We then used this platform for the discrimination of enterobacterial pathogens undistinguishable by MALDI-TOF, although leading to very different clinical outcomes. For each pathogen, we identified specific proteoforms that could potentially be used as biomarkers. We also improved the characterization of poorly described bacterial strains. Our results highlight the advantage of addressing proteoforms rather than peptides for accurate bacterial characterization and qualify top-down proteomics as a promising tool in clinical microbiology. Data are available via ProteomeXchange with the identifier PXD019247.