Search Thermo Fisher Scientific
Mass spectrometry-based Protein Footprinting
Protein Footprinting methods provide a means to explore protein structures and interactions by introducing covalent labels onto proteins. To identify modified amino acids and quantify the extent of labeling, researchers can use liquid chromatography coupled to high-resolution MS (LC-MS/MS).
In most cases, footprinting reports on the solvent accessibility of amino acid side chains, which is altered upon ligand binding or changes in conformation. Footprinting can be reversible as Hydrogen deuterium exchange (HDX), where deuterium in D2O replaces hydrogen of the backbone amides or irreversible/covalent labeling as Hydroxyl Radical Protein Footprinting (HRPF) or FPOP which utilizes hydroxyl (OH) radicals or CF3 to oxidatively modify the side chains of amino acids on the time scale of sub-milliseconds.
Coupling these footprinting methods with bottom-up proteomics, where proteins are proteolyzed and the resulting peptides are analyzed by MS, results in localized information on interaction sites and regions of conformational changes.
Thermo Scientific mass spectrometers enable deep coverage with accurate and precise quantitation of the modified amino acids at scale.
5-Step workflow for MS-based footprinting
The 5-step workflow provides information about the basic steps for MS Protein Footprinting.
Pillars of a Protein Footprinting platform
What you get from Thermo Scientific Protein Footprinting platform
Thermo Scientific Orbitrap-based MS footprinting offers a robust method for the analysis of protein conformation, dynamics, and protein-protein interactions.
Our footprinting platform detects and quantifies modified amino acids with sensitivity, dynamic range of analysis, accuracy of annotation, and reproducibility.
Accuracy of site localization
Precise localization of the modifications (oxidation) sites requires high quality mass spectra with accurate mass, exceptional resolution, and a complete series of fragment ions. Orbitrap-based mass spectrometers offer full m/z transmission of MS/MS scans to enable detection of the complete ion series for every type of fragmentation, to promote unambiguous annotation for site localization. Automatic gain control and normalized collision energy assure reproducible spectral quality within and across sample cohorts and enable robust quantification.
The Thermo Scientific Orbitrap Astral MS with its Astral analyzer MS/MS spectrum of oxidized peptide demonstrates the full sequence coverage of modified peptide and precise modification site localization (E6 vs E7).
Reproducibility
Validation of modification sites requires consistent performance and robust operation across cohorts of biological samples or technical replicates. The high reproducibility offered by high-resolution accurate mass (HRAM) Orbitrap MS, and liquid chromatography systems enables accurate comparisons across samples.
Thermo Scientific Vanquish Neo UHPLC Systems offer highly reproducible chromatographic separations and support a wide range of operations from nanoflow to high microflow.
Thermo Scientific Protein Footprinting workflow
Workflow for comprehensive discovery and characterization of modified (oxidized) amino acids, peptides
The Orbitrap mass spectrometry-based discovery workflow offers sensitivity across a full dynamic range of analysis, facilitating comprehensive detection and localization of labeling sites. To ensure high spectral quality for MS and MS/MS scans, which is crucial for accurate localization and quantification of labeled amino acids, this workflow utilizes meticulous sample preparation, highly reproducible chromatographic separations, and HRAM Orbitrap mass spectrometers. Moreover, the Thermo Scientific Orbitrap Ascend Structural Biology Tribrid Mass Spectrometer enables multiple fragmentation techniques: collision-induced dissociation [CID], higher-energy collisional dissociation [HCD] and electron transfer dissociation [ETD], MSn and can support single-level residue resolution for both chemical and radical footprinting by improving MS2 quality for site localization in the presence of multiple isomers.
Gain unparalleled sensitivity and dynamic range with the capability to detect peptides of varying abundance levels, whether high or low, in your sample. This is essential to comprehensively analyze both unmodified and modified peptides, allowing for the achievement of residue-level resolution.
What customers are saying about the Orbitrap Astral MS
"The sensitivity, mass accuracy, and speed of acquisition of the Orbitrap Astral MS enhances the breadth of Protein Footprinting in complex model systems, providing deep oxidation coverage. These capabilities facilitate the identification of low abundant modified amino acids ultimately leading to the possibility of utilizing this method for studying health disparities in primary patient samples."
Lisa M. Jones
Professor and Chancellor's Associates Endowed Chair in Chemistry and Biochemistry, University of California San Diego
What customers are saying about the Orbitrap Tribrid MS
"Hydroxyl radical Protein Footprinting (HRPF) at amino acid resolution is a very demanding LC-MS/MS experiment. Having the flexibility of multiple ion activation methods allows us to use CID or HCD for efficient fragmentation and assignment when we can chromatographically resolve oxidation isomers and have ETD fragmentation to quantify oxidation isomers when they co-elute. With the proper chromatography to ideally co-elute all oxidation isomers, we can even quantify all oxidation sites in a peptide by ETD with a single MS/MS spectrum for easier DDA method development."
Joshua Sharp
Triplett-Behrakis Professor of Pharmacology at the University of Mississippi and the Chief Technology Officer for GenNext Technologies, Inc.
For Research Use Only. Not for use in diagnostic procedures.