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Investigating alternative acidic proteases for H/D exchange coupled to mass spectrometry: Plasmepsin 2 but not plasmepsin 4 is active under quenching conditions

  • Julien Marcoux
  • Eric Thierry
  • Corinne Vivès
  • Luca Signor
  • Franck Fieschi
  • Eric ForestEmail author
Application Note

Abstract

Structural studies of proteins by hydrogen/deuterium exchange coupled to mass spectrometry (DXMS) require the use of proteases working at acidic pH and low temperatures. The spatial resolution of this technique can be improved by combining several acidic proteases, each generating a set of different peptides. Three commercial aspartic proteases are used, namely, pepsin, and proteases XIII and XVIII. However, given their low purity, high enzyme/protein ratios have to be used with proteases XIII and XVIII. In the present work, we investigate the activity of two alternative acidic proteases from Plasmodium falciparum under different pH and temperature conditions. Peptide mapping of four different proteins after digestion with pepsin, plasmepsin 2 (PSM2), and plasmepsin 4 (PSM4) were compared. PSM4 is inactive at pH 2.2 and 0°C, making it unusable for DXMS studies. However, PSM2 showed low but reproducible activity under DXMS conditions. It displayed no substrate specificity and, like pepsin, no strict sequence specificity. Altogether, these results show that PSM2 but not PSM4 is a potential new tool for DXMS studies.

Keywords

Plasmodium Falciparum Aspartic Protease Acidic Protease Bovine Hemoglobin Exchange Mass Spectrometry 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Supplementary material

13361_2011_210100076_MOESM1_ESM.doc (153 kb)
Supplementary material, approximately 157 KB.
13361_2011_210100076_MOESM2_ESM.pdf (2 mb)
Supplementary material, approximately 2114 KB.

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Copyright information

© American Society for Mass Spectrometry 2010

Authors and Affiliations

  • Julien Marcoux
    • 1
    • 2
  • Eric Thierry
    • 1
  • Corinne Vivès
    • 2
  • Luca Signor
    • 1
  • Franck Fieschi
    • 2
  • Eric Forest
    • 1
    Email author
  1. 1.Laboratoire de Spectrométrie de Masse des ProtéinesInstitut de Biologie StructuraleGrenobleFrance
  2. 2.Laboratoire des Protéines MembranairesUniversité Joseph Fourier, Institut de Biologie Structurale (IBS)GrenobleFrance

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