title: Investigating alternative acidic proteases for H/D exchange coupled to mass spectrometry: plasmepsin 2 but not plasmepsin 4 is active under quenching conditions.
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 degrees 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.
authors.affiliation: Laboratoire de Spectrométrie de Masse des Protéines, Institut de Biologie Structurale, Grenoble, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Marcoux
authors.forename: Eric
authors.initials: E
authors.lastname: Thierry
authors.forename: Corinne
authors.initials: C
authors.lastname: Vivès
authors.forename: Luca
authors.initials: L
authors.lastname: Signor
authors.forename: Franck
authors.initials: F
authors.lastname: Fieschi
authors.forename: Eric
authors.initials: E
authors.lastname: Forest
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D041961:Deuterium Exchange Measurement; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism; D006736:Horses; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D010434:Pepsin A / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D006454:Hemoglobins; D009211:Myoglobin; D011506:Proteins; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; D010434:Pepsin A; C089643:plasmepsin