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Metalloproteome factory: from crude extract to identification of metalloproteins via 3-dimensional HPLC-ICP-sfMS

ABSTRACT: Metalloproteins are essential for a vast array of biological processes in all living organisms. Identification of multiple metalloproteins from native expression (“metalloproteomics”) based on a single chromatography has low resolution and/or sensitivity, while classical protein purification lacks the versatility required for purifying multiple proteins for proteomics. We present a new metalloproteomics method based on three dimensions of liquid chromatography hyphenated with inductively coupled sector-field mass spectrometry (ICP-sfMS): anion exchange chromatography (AEC) followed by either chromatofocusing (CF) or hydrophobic interaction chromatography (HIC), and finally size exclusion chromatography (SEC). We used this 3D technique to purify naturally abundant metalloproteins from crude (mainly) plant, bacteria and animal extracts to a quantity and purity sufficient for protein identification and quantification by mass spectrometry (Tims-TOF-MS). Sensitivity and signal/background ratio were sufficient to identify proteins at concentrations < 1 nM and total amounts < 1 pM in the final SEC.

INSTRUMENT(S):

ORGANISM(S): Trichodesmium Erythraeum Ims101 Glycine Max

TISSUE(S): Whole Body

SUBMITTER: Filip Dyčka

LAB HEAD: Hendrik Küpper

PROVIDER: PXD056024 | Pride | 2025-08-25

REPOSITORIES: Pride

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Publications

Phaco-vitrectomy for macular holes.

Kumudhan Dharmalingam D Simcock Peter R PR

Acta ophthalmologica Scandinavica 20051001 5

PMID: 16188014

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Comparison of EVs isolation methods for miRNAs sequencing

Project description:Background & aims: MicroRNAs (miRNAs) encapsulated in EVs are potential diagnostic and prognostic biomarkers. However, discrepancies on miRNA patterns and their validation are still frequent due to differences in sample origin, EVs isolation, miRNA extraction and sequencing methods. Selecting appropriate EVs isolation methods is therefore a critical step for miRNA-based biomarker discovery. The aim of the present study is to find the most suitable EVs isolation method for miRNAs sequencing adequate for clinical application. Material & Methods EVs were isolated by Size Exclusion Chromatography (SEC), iodixanol gradients (GRAD) and the combination of both (SEC+GRAD), using the same plasma sample, in triplicate isolation assays. Isolated EVs were characterized and RNA was extracted. Three different protocols for miRNA library preparation were compared (NEBNext, NEXTFlex and SMARTer smRNA-seq) and miRNAs encapsulated on EVs were sequenced using NextSeq 500 system (Illumina). Finally, the yield, abundance and diversity of miRNAs using the three different EVs isolation protocols were analyzed and compared between them. Results The majority of lipoproteins, total cholesterol and plasma proteins were removed from the EVs-containing fractions by using SEC, GRAD, and SEC+GRAD. SEC method recovered a larger amount of EVs followed by GRAD and SEC+GRAD, while GRAD and SEC+GRAD yielded the purest vesicles. NEBNext was the library preparation kit that showed the highest reproducibility among replicas, higher number of reads corresponding to miRNAs and more different miRNAs, followed by NEXTFlex and SMARTer smRNA-seq. GRAD method showed the highest reproducibility among replicas, a higher number of reads corresponding to miRNAs and more different miRNAs, followed by SEC and SEC+GRAD methods. Conclusions These results render the GRAD method to isolate EVs as one of the most appropriate to detect miRNAs from Evs.

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