Dataset Information

Arabidopsis thaliana leaf proteome label-free shotgun digested by Legumain at various pH

ABSTRACT: Proteomic optimisation of Arabidopsis thaliana leaf proteome generated using legumain (cleave C-terminal of asparagine/aspartic acid) as protease at different pH from pH 5.0 to pH 6.5.

INSTRUMENT(S): maXis

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Leaf

SUBMITTER: Fatih Demir

LAB HEAD: Pitter Florien Huesgen

PROVIDER: PXD014698 | Pride | 2020-01-20

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	Legumain_pH_MQ_txt.zip	Other
	Legumain_pH_Raw_Data.zip	Other

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Publications

ExteNDing Proteome Coverage with Legumain as a Highly Specific Digestion Protease.

Soh Wai Tuck WT Demir Fatih F Dall Elfriede E Perrar Andreas A Dahms Sven O SO Kuppusamy Maithreyan M Brandstetter Hans H Huesgen Pitter F PF

Analytical chemistry 20200131 4

Bottom-up mass spectrometry-based proteomics utilizes proteolytic enzymes with well characterized specificities to generate peptides amenable for identification by high-throughput tandem mass spectrometry. Trypsin, which cuts specifically after the basic residues lysine and arginine, is the predominant enzyme used for proteome digestion, although proteases with alternative specificities are required to detect sequences that are not accessible after tryptic digest. Here, we show that the human cy ...[more]

PMID: 31951383

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Project description:Gene expression profiles of Escherichia coli K-12 W3110 were compared as a function of steady-state external pH. Cultures were grown with aeration to an optical density at 600 nm of 0.3 in potassium-modified Luria-Bertani medium buffered at pH 5.0, 7.0, and 8.7. For each of the three pH conditions, cDNA from RNA of five independent cultures was hybridized to Affymetrix E. coli arrays. Analysis of variance with a significance level of 0.001 resulted in 98% power to detect genes showing a twofold difference in expression. Normalized expression indices were calculated for each gene and intergenic region (IG). Differential expression among the three pH classes was observed for 763 genes and 353 IGs. Hierarchical clustering yielded six well-defined clusters of pH profiles, designated Acid High (highest expression at pH 5.0), Acid Low (lowest expression at pH 5.0), Base High (highest at pH 8.7), Base Low (lowest at pH 8.7), Neutral High (highest at pH 7.0, lower in acid or base), and Neutral Low (lowest at pH 7.0, higher at both pH extremes). Flagellar and chemotaxis genes were repressed at pH 8.7 (Base Low cluster), where the cell's transmembrane proton potential is diminished by the maintenance of an inverted pH gradient. High pH also repressed the proton pumps cytochrome o (cyo) and NADH dehydrogenases I and II. By contrast, the proton-importing ATP synthase F1Fo and the microaerophilic cytochrome d (cyd), which minimizes proton export, were induced at pH 8.7. These observations are consistent with a model in which high pH represses synthesis of flagella, which expend proton motive force, while stepping up electron transport and ATPase components that keep protons inside the cell. Acid-induced genes, on the other hand, were coinduced by conditions associated with increased metabolic rate, such as oxidative stress. All six pH-dependent clusters included envelope and periplasmic proteins, which directly experience external pH. Overall, this study showed that (i) low pH accelerates acid consumption and proton export, while coinducing oxidative stress and heat shock regulons; (ii) high pH accelerates proton import, while repressing the energy-expensive flagellar and chemotaxis regulons; and (iii) pH differentially regulates a large number of periplasmic and envelope proteins. Keywords: Steady State