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Increasing protein charge state when using laser electrospray mass spectrometry.

Santosh Karki1, Paul M Flanigan, Johnny J Perez

  • 1Department of Chemistry and Center for Advanced Photonics Research, Temple University, Philadelphia, PA, 19122, USA.

Journal of the American Society for Mass Spectrometry
|March 11, 2015
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Summary

Femtosecond laser electrospray mass spectrometry (LEMS) enhances protein charge states. This method, using laser vaporization and supercharging reagents, significantly increases ion abundance in higher charge states for acid-sensitive proteins like cytochrome c and myoglobin.

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Area of Science:

  • Analytical Chemistry
  • Biochemistry
  • Mass Spectrometry

Background:

  • Electrospray ionization mass spectrometry (ESI-MS) is crucial for analyzing proteins.
  • Protein charge states in ESI-MS are influenced by solvent conditions and protein acid stability.
  • Conventional ESI-MS can be limited in its ability to generate high charge states for certain proteins.

Purpose of the Study:

  • To investigate the utility of femtosecond laser vaporization coupled with electrospray ionization (LEMS) for protein analysis.
  • To compare the charge state distributions of proteins analyzed by LEMS versus conventional ESI-MS.
  • To evaluate the impact of supercharging reagents and electrospray conditions on protein charge states.

Main Methods:

  • Femtosecond laser vaporization was employed to transfer proteins (cytochrome c, myoglobin, lysozyme, ubiquitin) into an electrospray plume.
  • A mixture of a supercharging reagent (m-nitrobenzyl alcohol) and acids (TFA, AA, FA) was used in the electrospray solution.
  • Laser electrospray mass spectrometry (LEMS) was performed and compared with conventional ESI-MS.
  • Measurements were conducted as a function of electrospray flow rate.

Main Results:

  • LEMS demonstrated a marked increase in ion abundance of higher charge states for acid-sensitive proteins (cytochrome c, myoglobin) compared to conventional ESI-MS.
  • Cytochrome c showed charge states from 19+ to 21+ with LEMS versus 13+ to 16+ with ESI-MS.
  • Myoglobin showed charge states from 19+ to 26+ with LEMS versus 18+ to 21+ with ESI-MS.
  • Increasing protein charge states (Zavg, Zmode) were observed for acid-sensitive proteins with LEMS.
  • Decreasing electrospray flow rates resulted in increasing charge states for cytochrome c and myoglobin.

Conclusions:

  • Femtosecond laser vaporization coupled with electrospray ionization (LEMS) is an effective technique for generating higher charge states in proteins.
  • LEMS offers advantages over conventional ESI-MS for analyzing acid-sensitive proteins by increasing ion abundance in higher charge states.
  • The method shows potential for improved protein characterization and analysis in mass spectrometry.