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Related Concept Videos

Electrospray Ionization (ESI) Mass Spectrometry01:12

Electrospray Ionization (ESI) Mass Spectrometry

Higher molecular weight biomolecules are nonvolatile compounds that may decompose before ionizing or vaporizing during mass analysis with conventional electron impact ionization methods. Accordingly, electrospray ionization (ESI) is the favored method for vaporizing and ionizing biomolecules as it circumvents rapid fragmentation and enables the recording of mass signals for the entire biomolecule.
ESI utilizes electrical energy to transfer ions from the liquid phase of the sample into the...
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Tandem mass spectrometry, also known as MS/MS or MS2, is an analytical technique that employs two mass analyzers. Essentially it is a series of mass spectrometers that helps isolate a particular biomolecule and then helps study its chemical properties.
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...

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Related Experiment Video

Updated: Jun 4, 2026

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins
12:47

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Published on: December 27, 2016

Serine/Threonine Phosphorylation Site Identification by ESI-MS.

Albert Sickmann, Helmut E Meyer

    CSH Protocols
    |March 2, 2011
    PubMed
    Summary
    This summary is machine-generated.

    This study presents a protocol to differentiate protein autophosphorylation from endogenous phosphorylation using radioactive ATP and mass spectrometry. This method aids in understanding signal transduction pathway regulation.

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    Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins
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    Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

    Published on: February 27, 2020

    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Cell Signaling

    Background:

    • Protein phosphorylation is crucial for cellular signaling, regulated by kinases and autophosphorylation.
    • Distinguishing between these phosphorylation types is vital for understanding signal transduction pathways.

    Purpose of the Study:

    • To develop a protocol for distinguishing protein autophosphorylation from endogenous phosphorylation.
    • To localize phosphorylation sites accurately within signaling proteins.

    Main Methods:

    • Incubation with radioactive ATP to label autophosphorylation sites.
    • Trypsin digestion of proteins into peptides.
    • Analysis using nano-high-performance liquid chromatography (nano-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS).
    • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation.

    Main Results:

    • The protocol successfully differentiates autophosphorylation from endogenous phosphorylation.
    • Localization of phosphorylation sites is achieved with high sensitivity.
    • The method requires minimal protein input (20 pmol) and detects low stoichiometry (<5%).

    Conclusions:

    • This protocol provides a robust method for site-specific phosphorylation analysis.
    • It enhances the understanding of signal transduction mechanisms regulated by autophosphorylation.
    • The technique is applicable to various proteins involved in cellular signaling.