Dataset Information

ASAP – Automated Sonication free Acid assisted Proteomes from cells & FFPE tissues

ABSTRACT: Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for retrospective studies but protein extraction and subsequent sample processing steps have shown to be challenging for mass spectrometry (MS) analysis. Streamlined high-throughput sample preparation workflows are essential for efficient peptide extraction from complex clinical specimens such as fresh frozen tissues or FFPE. Overall, proteome analysis has gained significant improvements in the instrumentation, acquisition methods, sample preparation workflows and analysis pipelines yet even the most recent FFPE workflows remain complex and are not readily scalable. Here, we present an optimized workflow for Automated Sonication-free Acid-assisted Proteome (ASAP) extraction from FFPE sections. ASAP enables efficient protein extraction from FFPE specimens achieving similar proteome coverage as established methods using time in equipment-heavy sonication-based methods at reduced sample processing time. The broad applicability of ASAP on archived pediatric tumor FFPE specimens resulted in high-quality data with increased proteome coverage and quantitative reproducibility. Our study demonstrates the practicality and superiority of the ASAP workflow as a streamlined, time and cost-effective pipeline for high-throughput FFPE proteomics of clinical specimens.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Whole Body

SUBMITTER: Philipp Lange

LAB HEAD: Philipp Lange

PROVIDER: PXD036910 | Pride | 2024-05-21

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	GB_VG_20220226_25cmPepSep_notrap_ffpe-TFA_2.mgf	Mgf
	GB_VG_20220226_25cmPepSep_notrap_ffpe-TFA_2.raw	Raw
	GB_VG_20220226_25cmPepSep_notrap_ffpe-TFA_4.mgf	Mgf
	GB_VG_20220226_25cmPepSep_notrap_ffpe-TFA_4.raw	Raw
	GB_VG_20220226_25cmPepSep_notrap_ffpe-TFE_1.mgf	Mgf

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Publications

ASAP─Automated Sonication-Free Acid-Assisted Proteomes─from Cells and FFPE Tissues.

Barnabas Georgina D GD Goebeler Verena V Tsui Janice J Bush Jonathan W JW Lange Philipp F PF

Analytical chemistry 20230201 6

Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for retrospective studies, but protein extraction and subsequent sample processing steps have been shown to be challenging for mass spectrometry (MS) analysis. Streamlined high-throughput sample preparation workflows are essential for efficient peptide extraction from complex clinical specimens such as fresh frozen tissues or FFPE. Overall, proteome analysis has gained significant improvements in the instrumentation, acq ...[more]

PMID: 36724070

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Project description:As proteins represent cell function, proteomic analysis of prostate tissues may elucidate the mechanisms of prostate cancer (PCa) progression. To that end, formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources, but associated with large analytical difficulties due to protein cross-linking. Our aim is to characterize tissue protein changes associated with castration resistant PCa. This study focused at the development of a reliable protocol for the proteomic analysis of FFPE tissues which was followed by a pilot study using FFPE PCa clinical samples to investigate whether the optimized protocol can provide biologically relevant data in the context of PCa. Archival FFPE mouse tissues were processed using seven protein extraction protocols including combinations of homogenization means (beads, sonication, boiling) and buffers (Tris-HCl and Urea-Thiourea). The proteomics output was evaluated by SDS electrophoresis and high resolution LC-MS/MS analysis. FFPE tissues (3 sections, 15��m each per sample) from 10 patients with PCa corresponding to tumor (GS=6 or GS��8) and adjacent benign regions were processed with the optimized protocol. Extracted proteins were analyzed by GeLC-MS/MS followed by statistical and bioinformatics analysis using Cytsoscape and Open Targets tool. A combination of cell lysis by Tris-HCl, SDS and DTE with bead homogenization, sonication and boiling provided most efficient protein extraction from FFPE tissues based on protein identifications and reproducibility. Comparison between the FFPE and matched FF tissues showed a substantial overlap in protein identifications (502 common out of 1,216 IDs commonly identified in all FF samples and 530 IDs commonly identified in all FFPE samples) with a strong correlation in relative abundances (rs=0.846, p<.001). Proteins significantly deregulated between PCa GS��8 and PCa GS=6 represented extracellular matrix organisation, phosphorylation and gluconeogenesis pathways, while proteins deregulated between cancerous tissues and benign counterparts, reflected increased translation, peptide synthesis and protein metabolism in the former, as expected. Further disease association analysis using the Open Targets platform showed that 60% of the differentially expressed proteins in cancer versus normal adjacent were significantly associated with PCa. These results support the relevance of the proteomic findings in the context of PCa and the reliability of the optimized protocol for proteomics analysis of FFPE material. A large scale study including 160 clinical FFPE prostate cancer and benign tissues to define protein changes associated with aggressive PCa, is planned.