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Mass spectrometry-based crosslinking analysis
Gain a better understanding of how interactions influence biological processes in cells with crosslinking mass spectrometry (XL-MS), which analyzes protein-protein and protein-nucleic acid interactions that are "locked in place." The functions of most proteins depend on their interactions with other proteins and cellular components, including nucleic acids and fatty acids. It is through these interactions that biological processes begin, evolve, and conclude.
Obtain localized information about interaction sites and areas of conformational change by coupling chemical labeling with bottom-up proteomics, which involves the proteolysis of proteins and subsequent analysis of the resulting peptides by mass spectrometry. Thermo Scientific mass spectrometers provide the deep coverage necessary for robust analysis, ensuring accurate and precise quantitation of crosslinked residues at an unprecedented scale.
Benefits of the Thermo Scientific Crosslinking MS platform
Thermo Scientific Orbitrap-based crosslinking-MS offers a robust method for the analysis of protein conformation, protein-protein and protein-nucleic acid interactions.
Our crosslinking-MS platform provides sensitivity, dynamic range of analysis, accuracy of annotation, and robust quantification of crosslinked peptides.
Selection of crosslinkers
Protein crosslinking
Crosslinking reagents are molecules that chemically attach to specific functional groups of proteins, creating distance constraints for structural mapping.
When choosing crosslinkers for various applications, several factors should be considered: chemical specificity, spacer arm length, water solubility, and cell membrane permeability. Thermo Fisher Scientific offers a wide range of crosslinking reagents and a Crosslinker Selection Tool to assist in this process.
Protein-nucleic acid crosslinking
RNA/DNA-protein complexes play a crucial role in various cellular processes, including DNA replication, DNA repair, transcription, splicing, RNA maturation, and translation control.
The linkages between protein and RNA/DNA can be induced by UV light or chemical reagents. UV light crosslinks various amino acids primarily to uracil, while chemical methods mainly connect amino acids to guanosine and adenosine.
Sensitivity and dynamic range of detection
Crosslinked peptides are usually present in relatively low abundance in complex samples that exhibit a wide dynamic range of peptide intensities. Thermo Scientific Orbitrap-based mass spectrometers can support the simultaneous detection of very high and very low-level analytes.
Site localization accuracy
Precise localization of crosslinking sites requires high-quality mass spectra with accurate mass, exceptional resolution, and a complete series of fragment ions.
Orbitrap-based mass spectrometers provide full m/z transmission of MS/MS scans, allowing for the detection of the entire ion series for all types of fragmentation. They also facilitate the identification of immonium ions without discrimination, which aids in unambiguous site localization. The use of automatic gain control (AGC) and normalized collision energy optimizes the detection for crosslinked peptides, ensuring reproducible spectral quality for reliable and robust quantification.
Thermo Scientific Orbitrap Astral MS with its Astral analyzer MS/MS spectrum of crosslinked peptide demonstrates the full sequence coverage and precise crosslinked sites localization.
Thermo Scientific protein crosslinking workflow
Thermo Scientific Orbitrap mass spectrometry-based discovery workflow provides sensitivity for a full dynamic range of analysis to enable comprehensive crosslinked sites detection and localization. The discovery workflow leverages sample preparation, highly reproducible chromatographic separations and high-resolution, accurate mass (HRAM) Orbitrap mass spectrometers that provide high spectral quality for both MS and MS/MS scans, necessary for confident localization and quantification of crosslinks.
Moreover, Thermo Scientific Orbitrap Ascend Structural Biology Tribrid Mass Spectrometer enables Real-Time Library Search triggering of high-resolution MS2 scans based on the intensity ratios, significantly enhancing crosslinked precursors detection. The approach particularly improves productivity for higher throughput and resource-constrained biological projects.
Monolinks complicate the identification of crosslinked peptides by MS, even after enrichment. By analyzing the intensity ratios of specific peaks associated with enrichable crosslinkers, it is possible to partially distinguish between crosslinks and monolinks in samples.
Real-Time Library Search activates high-resolution MS2 scans based on these intensity ratios. This feature effectively removes monolinks and unmodified peptides, significantly improving the detection of cross-linked species.
What customers are saying about Orbitrap Tribrid MS
"The unique feature of Real-Time Library Search on the Orbitrap Ascend Structural Biology Tribrid Mass Spectrometer enables on-the-fly selection of crosslinked precursors for MS2 sequencing. This is really exciting and has always been what we hoped to achieve over the last years—selective targeting and sequencing of cross-linked peptides."
Fan Liu, PhD
Professor of Biochemistry
Leibniz Research Institute for Molecular Pharmacology
Thermo Scientific protein-nucleic acid crosslinking workflow
Thermo Scientific mass spectrometers offer robust, sensitive, and reproducible solutions for protein-nucleic acid crosslinking workflow. The Orbitrap Astral Mass Spectrometer and Thermo Scientific Orbitrap Exploris series of mass spectrometers series offer a wide dynamic range along with high-resolution, accurate mass precision for both identification and quantitation.
The NuXL node, embedded in Thermo Scientific Proteome Discoverer Software, facilitates the analysis of UV or chemically induced protein-RNA/DNA crosslinking data. Furthermore, label-free or TMT quantitation of crosslinked peptide-RNA/DNA oligonucleotide can be achieved accurately and reproducibly using precursor or reporter ion quantification.
Astral analyzer MS/MS spectrum of crosslinked peptide-RNA oligonucleotide. Also showing the reporter ion region used for quantitation.
Example of TMT quantitation of UV crosslinked E.coli ribosomes in triplicates at a ratio of 2:1, demonstrating accurate and precise quantitation.
"The protein-nucleic acid crosslinking workflow combines crosslinking, enzymatic digestion, enrichment, mass spectrometric analysis, and final NuXL-based data evaluation. It can be applied to any protein-RNA/DNA complex as well as to entire cells. With the NuXL node implemented in Proteome Discoverer Software, we are able to detect and analyze protein-RNA/DNA interactions, along with annotation and even quantification at the amino acid level."
Professor Henning Urlaub
Max Planck Institute for Multidisciplinary Sciences
University Medical Center Göttingen
For Research Use Only. Not for use in diagnostic procedures.