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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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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Project description:Gliadin triggers T-cell mediated immunity in celiac disease, and has cytotoxic effects on enterocytes mediated through obscure mechanisms. In addition, gliadin transport mechanisms, potential cell surface receptors and gliadin-activated downstream signaling pathways are not completely understood. In order to screen for novel downstream gliadin target genes we performed a systematic whole genome expression study on intestinal epithelial cells. Undifferentiated Caco-2 cells were exposed to pepsin- and trypsin- digested gliadin (PT-G), a blank pepsin-trypsin control (PT) and to a synthetic peptide corresponding to gliadin p31-43 peptide for six hours. RNA from four different experiments was used for hybridization on Agilent one color human whole genome DNA microarray chips. The microarray data were analyzed using the Bioconductor package LIMMA. Genes with nominal p < 0.01 were considered statistically significant. Compared to the untreated cells 1705, 1755 and 211 probes were affected by PT-G, PT and p31-43 respectively. 46 probes were significantly different between PT and PT-G treated cells. Among the p31-43 peptide affected probes, 10 and 21 probes were affected by PT-G and PT respectively. Only PT-G affected genes could be validated by quantitative real-time polymerase chain reaction. All the genes were, nonetheless, also affected to a comparable level by PT treated negative controls. In conclusion, we could not replicate previously reported direct effects of gliadin peptides on enterocytes. The PT-G affected genes in the microarray analysis were validated by qRT-PCR, however these genes were also affected by PT treated negative controls suggesting that certain epitopes derived from pepsin and trypsin may also affect epithelial cell gene transcription. Our study demonstrates novel non-enzymatic effects of pepsin and trypsin on cells and calls for proper controls in pepsin and trypsin digested gliadin experiments. It is conceivable that gliadin effects on enterocytes are secondary mediated through oxidative stress, NFkB activation and IL-15 up-regulation. In total, 16 samples were analyzed of which 4 were control (MED) samples, 4 samples of p31-43 treatment, 4 samples of PT treatmetn and 4 samples of PT-G treatment

Dataset Information

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

Publications

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

Similar Datasets

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