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Related Concept Videos

Mass Spectrometry: Isotope Effect01:13

Mass Spectrometry: Isotope Effect

Most elements exist in nature as a mixture of isotopes. The isotopes differ in weight due to their respective number of neutrons. The molecular weight of a molecule is different depending on the specific isotope of its elements involved. As a result, the mass spectrum of the molecule exhibits peaks from the same fragment at multiple positions. The positions of these mass signals depend on the mass differences between isotopes. Furthermore, the intensity of these signals is dependent on the...
MALDI-TOF Mass Spectrometry01:19

MALDI-TOF Mass Spectrometry

Mass spectrometry is a powerful characterization technique that can identify and separate a wide variety of compounds ranging from chemical to biological entities, based on their mass-to-charge ratio (m/z). The instruments that allow this detection, known as mass spectrometers, have three components: an ion source, a mass analyzer, and a detector. These spectrometers differ based on the nature of their ion source and analyzers.Matrix-assisted laser desorption ionization (MALDI) is a commonly...
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Matrix-Assisted Laser Desorption Ionization (MALDI)

Matrix-assisted laser desorption ionization (MALDI) is a powerful analytical technique used in mass spectrometry. It enables the identification and characterization of various biomolecules, including proteins, peptides, nucleic acids, and carbohydrates. MALDI is an ionization technique, widely employed in biological and medical research, as well as in fields like pharmacology and biochemistry.The analyte of interest, a biomolecule or a mixture of biomolecules, is mixed with a suitable matrix...
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Tandem Mass Spectrometry01:21

Tandem Mass Spectrometry

Tandem mass spectrometry is a technique that uses multiple mass analyzers in series to obtain a higher selectivity and reduce chemical noise during analyte detection. Instruments with multiple analyzers separated by an interaction cell enable secondary fragmentation and selected study of the fragment ions.Secondary fragmentations occur in the interaction cell and can be induced by various factors. Fragmentation induced by collision with inert gases, such as N2, Ar, He, etc., is called...
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Inductively coupled plasma–mass spectrometry (ICP–MS) is a highly selective and sensitive technique for accurate elemental analysis. Though the analysis of ICP–MS mass spectra is comparatively straightforward, it is affected by spectroscopic and non-spectroscopic interferences. Spectroscopic interferences arise when the plasma contains ionic species with an m/z value the same as the analyte ion. Spectroscopic interference can be categorized as isobaric, polyatomic ions, and refractory oxide ion...

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Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer
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Published on: November 26, 2013

Direct Competitive Kinetic Isotope Effect Measurement Using Quantitative Whole Molecule Matrix-Assisted Laser

Teodora Kljaic1, Merritt A Scott1, Veronica Guirguis1

  • 1Department of Chemistry & Biochemistry, University of Maryland at College Park, College Park, Maryland, USA.

Chembiochem : a European Journal of Chemical Biology
|June 24, 2026
PubMed
Summary
This summary is machine-generated.

We developed a new mass spectrometry method for directly measuring enzyme reaction kinetics. This technique simplifies kinetic isotope effect (KIE) analysis, offering precise insights into enzyme mechanisms.

Keywords:
β‐galactosidaseenzymologykinetic isotope effectmass spectrometrymatrix‐assisted laser desorption ionization time‐of‐flight

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Area of Science:

  • Biochemistry
  • Enzymology
  • Analytical Chemistry

Background:

  • Kinetic isotope effect (KIE) measurements are crucial for understanding enzyme mechanisms and transition states.
  • Current KIE measurement methods are often complex and time-consuming, limiting their widespread application.
  • Direct measurement of competitive KIEs in enzymatic reactions requires robust analytical techniques.

Purpose of the Study:

  • To develop a simplified and direct method for measuring competitive kinetic isotope effects (KIEs) in enzyme-catalyzed reactions.
  • To apply matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for precise KIE determination.
  • To investigate the hydrolysis of lactose by E. coli β-galactosidase (LacZ) using the developed method.

Main Methods:

  • Developed a novel method utilizing whole molecule MALDI-TOF MS for direct KIE measurement.
  • Incorporated an isotope-labeled internal standard added at reaction quench points.
  • Simultaneously measured relative isotope ratios (R) and fractional conversion (F) over time.
  • Applied the method to determine KIEs for [1'-13C]lactose and [6'-13C]lactose in E. coli β-galactosidase catalyzed hydrolysis.

Main Results:

  • Successfully implemented a direct MALDI-TOF MS approach for competitive KIE measurements.
  • Achieved precise KIE measurements comparable to established radioisotope labeling and NMR methods.
  • Quantified KIEs for specific carbon isotopes ([1'-13C] and [6'-13C]) in lactose hydrolysis.
  • Demonstrated the method's utility for studying E. coli β-galactosidase kinetics.

Conclusions:

  • The developed MALDI-TOF MS method offers a direct, precise, and efficient way to measure enzymatic KIEs.
  • This technique simplifies the analytical workflow, making KIE studies more accessible.
  • The approach provides valuable insights into the microscopic steps of enzyme-catalyzed reactions, aiding in transition state analysis.