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Electrospray Ionization (ESI) Mass Spectrometry

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

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Published on: December 27, 2016

Histidine Phosphorylation Site Identification by ESI-MS.

Albert Sickmann, Helmut E Meyer

|March 2, 2011

Summary

Phosphohistidine is unstable in acidic conditions but stable on C(18) resins. This stability allows for analysis using HPLC and ESI-MS, overcoming limitations of traditional MALDI-MS methods.

Area of Science:

Biochemistry
Analytical Chemistry
Mass Spectrometry

Background:

Phosphohistidine is a phosphoamino acid with limited stability under acidic conditions (pH <5), degrading rapidly.
Traditional MALDI-MS methods using acidic matrices like α-cyano-4-hydroxycinnamic acid are unsuitable for phosphohistidine detection.
Alkaline MALDI matrices can reduce detection sensitivity for phosphorylated amino acids.

Purpose of the Study:

To investigate the stability of phosphohistidine under various conditions.
To develop a reliable method for the detection and analysis of phosphohistidine.
To overcome the limitations of existing mass spectrometry techniques for phosphohistidine analysis.

Main Methods:

Investigated phosphohistidine stability under acidic and alkaline conditions.

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Evaluated the use of C(18) column resins for phosphohistidine storage.

Employed High-Performance Liquid Chromatography (HPLC) with an acidic solvent system.

Utilized Electrospray Ionization Mass Spectrometry (ESI-MS) for detection.

Main Results:

Phosphohistidine exhibits a short half-life (<20 min) under acidic conditions (pH <5).
Phosphohistidine is surprisingly stable when stored on C(18) column resins, even under acidic conditions.
HPLC separation using C(18) resin and an acidic solvent system is feasible for phosphohistidine analysis.

Conclusions:

Rapid processing of proteins containing phosphohistidine under alkaline conditions is recommended.
C(18) column resins provide a means to stabilize phosphohistidine for analysis.
HPLC separation coupled with ESI-MS offers a viable alternative for detecting phosphohistidine, circumventing MALDI-MS limitations.