Dataset Information

Aspergillus niger Prolyl endoprotease (An-PEP) for Hydrogen-Deuterium Exchange Mass Spectrometry and Protein Structural Studies

ABSTRACT: To monitor the structural integrity of therapeutic proteins, hydrogen-deuterium exchange mass spectrometry (HDX-MS) is increasingly utilized in the pharmaceutical industry. The successful outcome of HDX-MS analyses depends on the sample preparation conditions, which involve the rapid digestion of proteins at 0Â°C and pH 2.5. Very few proteases are known to withstand such harsh conditions, with pepsin being the best-known exception, even though its activity is also strongly reduced at 0Â°C. Here, we evaluate the usage of a Prolyl-endopeptidase from Aspergillus niger (An-PEP) for HDX-MS. What makes this protease very attractive is its ability to cleave the hardest to digest amino acid, proline. To our surprise, and in contrast to previous reports, An-PEP activity was found optimal around pH 2.5 and could be further enhanced by urea up to 40%. Under typical HDX-MS conditions and using small amounts of enzyme, An-PEP generated an equivalent number of peptides as pepsin, as exemplified by using the two model systems; tetrameric human hemoglobin (Hb) and human IgG4. Interestingly, because An-PEP peptides are shorter than pepsin-generated peptides, higher sequence resolution could be achieved, especially for Pro-containing protein regions in the alpha subunit of Hb, revealing new protected Hb regions that were not observed with pepsin. Due to its Pro-preference and resistance to low pH, we conclude that An-PEP is an archetype enzyme for HDX-MS, highly complementary to pepsin, and especially promising for structural studies on Pro-rich proteins (PRPs) or proteins containing Pro-rich binding domains involved in cellular signaling.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Escherichia Coli

SUBMITTER: Liana Tsiatsiani

LAB HEAD: Albert JR Heck

PROVIDER: PXD006766 | Pride | 2017-07-12

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	OR9_LT_edp_ph2.5_HCD1.msf	Msf
	OR9_LT_edp_ph2.5_HCD1.raw	Raw
	OR9_LT_edp_ph2.5_HCD1_proteingroups.txt	Txt
	OR9_LT_edp_ph2.5_HCD1_psms.txt	Txt
	OR9_LT_edp_ph2.5_HCD1_searchinput.txt	Txt

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Aspergillus niger Prolyl Endoprotease for Hydrogen-Deuterium Exchange Mass Spectrometry and Protein Structural Studies.

Tsiatsiani Liana L Akeroyd Michiel M Olsthoorn Maurien M Heck Albert J R AJR

Analytical chemistry 20170712 15

To monitor the structural integrity of therapeutic proteins, hydrogen-deuterium exchange mass spectrometry (HDX-MS) is increasingly utilized in the pharmaceutical industry. The successful outcome of HDX-MS analyses depends on the sample preparation conditions, which involve the rapid digestion of proteins at 0 °C and pH 2.5. Very few proteases are able to withstand such harsh conditions, with pepsin being the best-known exception, even though its activity is also strongly reduced at 0 °C. Here, ...[more]

PMID: 28657298

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