Dataset Information

SP3 protocol for proteomic plant sample preparation prior LC-MS/MS

ABSTRACT: Quantitative protein extraction from biological samples, as well as contaminants removal before LC-MS/MS, is fundamental for the successful bottom-up proteomic analysis. Four sample preparation methods, including the ﬁlter-aided sample preparation (FASP), two single-pot solid-phase-enhanced sample preparations (SP3) on carboxylated or HILIC paramagnetic beads, and protein suspension trapping method (S-Trap) were evaluated for SDS removal from Arabidopsis thaliana (AT) lysate. Finally, the optimized carboxylated SP3 workflow was benchmarked closely against the routine FASP. Ultimately, LC-MS/MS analyses revealed that regarding the number of identifications, number of missed cleavages, proteome coverage, repeatability, reduction of handling time, and cost per assay, the SP3 on carboxylated magnetic particles proved to be the best alternative for SDS and other contaminants removal from plant sample lysate. A robust and efficient two-hour SP3 protocol for a wide range of protein input is presented, benefiting from no need to adjust amount of beads, binding and rinsing conditions, or digestion parameters.

INSTRUMENT(S): Q Exactive HF, LTQ Orbitrap Elite

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

TISSUE(S): Plant Cell, Leaf

SUBMITTER: Kamil Mikulasek

LAB HEAD: Zbyněk Zdráhal

PROVIDER: PXD022688 | Pride | 2021-02-26

REPOSITORIES: Pride

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SP3 Protocol for Proteomic Plant Sample Preparation Prior LC-MS/MS.

Mikulášek Kamil K Konečná Hana H Potěšil David D Holánková Renata R Havliš Jan J Zdráhal Zbyněk Z

Frontiers in plant science 20210310

Quantitative protein extraction from biological samples, as well as contaminants removal before LC-MS/MS, is fundamental for the successful bottom-up proteomic analysis. Four sample preparation methods, including the filter-aided sample preparation (FASP), two single-pot solid-phase-enhanced sample preparations (SP3) on carboxylated or HILIC paramagnetic beads, and protein suspension trapping method (S-Trap) were evaluated for SDS removal and protein digestion from <i>Arabidopsis thaliana</i> (A ...[more]

PMID: 33777071

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Project description:Understanding the interplay of the proteome and the metabolome aids in understanding cellular phenotypes. To enable more robust inferences from such multi-omics analyses, combining proteomic and metabolomic datasets from the same sample provides major benefits by reducing technical variation between extracts during the pre-analytical phase, decreasing sample variation due to varying cellular content between aliquots, and limiting the required sample amount. We evaluated the advantages, practicality and feasibility of a single-sample workflow for combined proteome and metabolome analysis. In the workflow, termed MTBE-SP3, we combined a fully automated protein lysis and extraction protocol (autoSP3) with a semi-automated biphasic 75% EtOH/MTBE extraction for quantification of polar/non-polar metabolites. Additionally, we compared the resulting proteome of various biological matrices (FFPE tissue, fresh-frozen tissue, plasma, serum and cells) between autoSP3 and MTBE-SP3. Our analysis revealed that the single-sample workflow provided similar results to those obtained from autoSP3 alone, with an 85-98% overlap of proteins detected across the different biological matrices. Additionally, it provides distinct advantages by decreasing (tissue) heterogeneity by retrieving metabolomics and proteomic data from the identical biological material, and limiting the total amount of required material. Lastly, we applied MTBE-SP3 to a lung adenocarcinoma cohort of 10 patients. Integrating the metabolic and proteomic alterations between tumour and non-tumour adjacent tissue yielded consistent data independent of the method used. This revealed mitochondrial dysfunction in tumor tissue through deregulation of OGDH, SDH family enzymes and PKM. In summary, MTBE-SP3 enables the facile and confident parallel measurement of proteins and metabolites obtained from the same sample. This workflow is particularly applicable for studies with limited sample availability and offers the potential to enhance the integration of metabolomic and proteomic datasets.

			Action	DRS
	Elite_FASP_AT_18h_1.raw	Raw
	Elite_FASP_AT_18h_2.raw	Raw
	Elite_FASP_AT_18h_3.raw	Raw
	Elite_FASP_AT_2_16h_1.raw	Raw
	Elite_FASP_AT_2_16h_2.raw	Raw