Dataset Information

Proteome profiling of Cumin by Q-Exactive HF mass spectrometer

ABSTRACT: A shotgun proteomic approach was used to obtain comprehensive dataset of cumin proteins. For this purpose we applied a one dimensional electrophoresis coupled with high resolution mass spectrometry. The first step in our proteome analysis strategy, we evaluate different extraction methods, based on differential solubility of protein. Among those methods, Readyprep protein extraction kit provide high purity of protein extract, gave higher protein yield and typically good resolution of protein bands on one dimensional gel electrophoresis. Further nanoLC-MS/MS analysis by state-of-the-art Q Exactive HF mass spectrometer resulted in identification of very high number proteins. Bioinformatics analysis provide functional distribution of identified protein. To our knowledge, this is the first study aimed for in depth study of cumin proteome.

INSTRUMENT(S): Q Exactive

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

SUBMITTER: Uzma Rana

LAB HEAD: Uzma Zaman

PROVIDER: PXD006257 | Pride | 2017-11-21

REPOSITORIES: Pride

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Publications

Protein Profiling of Non-model Plant Cuminum cyminum by Gel-Based Proteomic Approach.

Zaman Uzma U Urlaub Henning H Abbasi Atiya A

Phytochemical analysis : PCA 20171116 3

<h4>Introduction</h4>Cumin (Cuminum cyminum), a popular spice has been widely used in traditional medicine to cure various ailments. Despite the existence of scientific literature about its pharmacological properties, no successful proteome profiling has yet been attempted.<h4>Objective</h4>To optimise extraction of cumin proteins and analyse its profile by shotgun proteomics, using one-dimensional electrophoresis coupled with nano-ESI-LC-MS/MS.<h4>Methodology</h4>As a first step, we have compar ...[more]

PMID: 29148164

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Project description:Clavulanic acid (CLV) and amoxicillin, frequently administered in combination, can be independently involved in allergic reactions. Protein haptenation with β-lactams is considered necessary to activate the immune system. The aim of this study was to assess the suitability of biotinylated analogues of CLV as probes to study protein haptenation by this β-lactam. Two synthetic approaches afforded the labelling of CLV through esterification of its carboxylic group with a biotin moiety, via either direct binding (CLV-B) or tetraethylenglycol linker (CLV-TEG-B). The second analogue offered advantages as solubility in aqueous solution and potential lower steric hindrance for both intended interactions, with the protein and with avidin. NMR reactivity studies showed that both CLV and CLV-TEG-B reacts through β-lactam ring opening by aliphatic amino nitrogen, however with different stability of resulting conjugates. Unlike CLV conjugates, that promoted the decomposition of clavulanate fragment, the conjugates obtained with the CLV-TEG-B remained linked, as a whole structure including biotin, to nucleophile and showed a better stability. This was a desired key feature to allow CLV-TEG-B conjugated protein detection at great sensitivity. We have used biotin detection and mass spectrometry (MS) to detect the haptenation of human serum albumin (HSA) and human serum proteins. MS of conjugates showed that HSA could be modified by CLV-TEG-B. Remarkably, HSA preincubation with CLV excess only reduced moderately the incorporation of CLV-TEG-B, which could be attributed to different protein interferences. The CLV-TEG-B fragment with opened β-lactam was detected bound to the 404-430HSA peptide of the treated protein. Incubation of human serum with CLV-TEG-B resulted in the haptenation of several proteins that were identified by 2D-electrophoresis and peptide mass fingerprinting as HSA, haptoglobin, and heavy and light chains of immunoglobulins. Taken together, our results show that tagged-CLV keeps some of the CLV features. Moreover, although we observe a different behaviour in the conjugate stability and in the site of protein modification, the similar reactivity indicates that it could constitute a valuable tool to identify protein targets for haptenation by CLV with high sensitivity to get insights into the activation of the immune system by CLV and mechanisms involved in β-lactams allergy.

			Action	DRS
	Cumin_HF_Kit_1.raw	Raw
	Cumin_HF_Kit_10.raw	Raw
	Cumin_HF_Kit_11.raw	Raw
	Cumin_HF_Kit_2.raw	Raw
	Cumin_HF_Kit_3.raw	Raw