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Related Concept Videos

Mass Spectrometry: Complex Analysis01:21

Mass Spectrometry: Complex Analysis

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Mass spectrometry is an important technique for the identification of pure compounds. However, it has some limitations for the analysis of complex mixtures, often due to excessive fragmentation making the spectrum too complicated to decipher. Mass spectrometry can be combined with suitable separation methods in sequence, forming hyphenated methods, which are useful in the analysis of complex mixtures.
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Tandem mass spectrometry is a technique that uses multiple mass analyzers in series to obtain a higher selectivity and signal-to-noise ratio for the analyte. Instruments with multiple analyzers separated by an interaction cell enable secondary fragmentation and selected study of the fragment ions.
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The quadrupole mass analyzer consists of four cylindrical metal rods arranged in a diamond carrying a DC voltage and a radio-frequency AC voltage. The motion of ions through the quadrupole depends on the field strength, causing only ions of a certain m/z to resonate successfully and strike the detector at a given field strength. Though the transmission rate for these analyzers is high, the exact elemental composition of the sample is not determined because of low resolution; however, they are...
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The mass analyzer is a crucial component of the mass spectrometer. In the ionization chamber, the vaporized sample is bombarded with a high-energy electron beam to generate a radical cation and further fragment into neutral molecules, radicals, and cations. A series of negatively charged accelerator plates accelerate the cations into the mass analyzer. The mass analyzer separates ions according to their mass-to-charge (m/z) ratios and then directs them to the detector. The common types of mass...
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The resolution of a mass spectrometer depends on the efficiency of separating ions with different ion masses. The mass of an atom is approximated to the sum of the masses of protons and neutrons inside, considering the masses of protons and neutrons as equal. However, the masses of the proton (1.6726 × 10−24 g) and neutron (1.6749 × 10−24 g) are not truly equal. There is a minor error in the expression of atomic masses relative to the simplest atom of hydrogen. For...
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Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics
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Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition

Frantisek Filandr1, Vladimir Sarpe1, Shaunak Raval1,2

  • 1Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, T2N 4N1, Canada.

Nature Communications
|March 12, 2024
PubMed
Summary

We developed a new hydrogen/deuterium exchange method (HX-MS2) for faster, more accurate protein analysis. This technique improves data reliability and speeds up complex sample characterization, enabling wider applications in proteomics.

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Area of Science:

  • Proteomics
  • Biochemistry
  • Analytical Chemistry

Background:

  • Hydrogen/deuterium exchange (HX) coupled with mass spectrometry (MS) is a powerful technique for studying protein structure and dynamics.
  • Accurate deuterium incorporation calculations and peptide identification are crucial for reliable HX-MS data analysis.
  • Current methods can be time-consuming and require manual validation, limiting throughput and application scope.

Purpose of the Study:

  • To introduce a novel HX workflow coupled to tandem mass spectrometry (HX-MS2) for enhanced data acquisition and analysis.
  • To enable true auto-curation of HX data through the simultaneous acquisition of peptide precursors and fragment ions.
  • To validate the utility of HX-MS2 for high-throughput, proteomics-grade analysis of complex biological samples.

Main Methods:

  • Development of a hydrogen/deuterium exchange workflow integrated with tandem mass spectrometry (HX-MS2).
  • Utilized data-independent acquisition (DIA) methods compatible with most mass spectrometry platforms.
  • Employed collisional-induced dissociation (CID) to generate deuterated fragments for peptide identification and deuterium calculation authentication.
  • Applied a combinatorial strategy for confidence assessment of deuterium calculations based on fragment ion redundancy.

Main Results:

  • The HX-MS2 workflow enables simultaneous acquisition of peptide precursors and fragment ions, facilitating true auto-curation.
  • Deuterated fragments generated by CID confirm peptide identity and authenticate MS1-based deuterium calculations with high confidence.
  • The method significantly reduces analysis time and increases throughput for complex samples.
  • Demonstrated the application of HX-DIA in drug binding analysis of the protein kinase DNA-PKcs.

Conclusions:

  • HX-MS2 provides a robust, proteomics-grade approach for hydrogen/deuterium exchange mass spectrometry.
  • The auto-curation capability and high throughput of HX-MS2 streamline complex sample characterization.
  • This method offers a straightforward switch for laboratories utilizing DIA, promoting widespread adoption and application in various biological studies.