Accessing the reproducibility and specificity of pepsin and other aspartic proteases.
The aspartic protease pepsin is less specific than other endoproteinases. Because aspartic proteases like pepsin are active at low pH, they are utilized in hydrogen deuterium exchange mass spectrometry (HDX MS) experiments for digestion under hydrogen exchange quench conditions. We investigated the reproducibility, both qualitatively and quantitatively, of online and offline pepsin digestion to understand the compliment of reproducible pepsin fragments that can be expected during a typical pepsin digestion. The collection of reproducible peptides was identified from >30 replicate digestions of the same protein and it was found that the number of reproducible peptides produced during pepsin digestion becomes constant above 5-6 replicate digestions. We also investigated a new aspartic protease from the stomach of the rice field eel (Monopterus albus Zuiew) and compared digestion efficiency and specificity to porcine pepsin and aspergillopepsin. Unique cleavage specificity was found for rice field eel pepsin at arginine, asparagine, and glycine. Different peptides produced by the various proteases can enhance protein sequence coverage and improve the spatial resolution of HDX MS data. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.
Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA.
Joomi
J
Ahn
Min-Jie
MJ
Cao
Ying Qing
YQ
Yu
John R
JR
Engen
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism; D057055:Aspartic Acid Proteases / Q000737:chemistry* / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D004524:Eels; D005998:Glycine / Q000737:chemistry / Q000378:metabolism; D006736:Horses; D006859:Hydrogen / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D015203:Reproducibility of Results; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013552:Swine
D010455:Peptides; D001216:Asparagine; D006859:Hydrogen; D001120:Arginine; D003903:Deuterium; D057055:Aspartic Acid Proteases; D010434:Pepsin A; D005998:Glycine