Dataset Information

Automated sample preparation with SP3 for low-input clinical proteomics

ABSTRACT: High-throughput and streamlined workflows are essential in clinical proteomics for standardized processing of samples originating from a variety of sources, including frozen tissue, FFPE tissue, or blood. To reach this goal, we have implemented single-pot solid-phase-enhanced sample preparation (SP3) on a liquid handling robot for automated processing (autoSP3) of tissue lysates in a 96-well format, performing unbiased protein purification and digestion delivering peptides that can be directly analyzed by LCMS. AutoSP3 eliminates hands-on time and minimizes the risk of error, and we show it reduces the protein quantification variability, and improves longitudinal performance and reproducibility. We demonstrate the distinguishing ability of autoSP3 to process low-input samples, reproducibly quantifying 500-1000 proteins from 100-1000 cells (<100 ng protein). Furthermore we applied it to process a cohort of clinical FFPE pulmonary adenocarcinoma (ADC) samples, and recapitulate their separation into known histological growth patterns based on proteome profiles. Collectively, autoSP3 provides a generic, scalable, and cost-effective pipeline for routine and standardized proteomic sample processing that should enable reproducible proteomics in broad range of clinical and non-clinical applications.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Lung, Epithelial Cell, Hela Cell

DISEASE(S): Non-small Cell Lung Carcinoma

SUBMITTER: Torsten Mueller

LAB HEAD: Jeroen Krijgsveld

PROVIDER: PXD014556 | Pride | 2020-01-10

REPOSITORIES: Pride

ACCESS DATA

Publications

Automated sample preparation with SP3 for low-input clinical proteomics.

Müller Torsten T Kalxdorf Mathias M Longuespée Rémi R Kazdal Daniel N DN Stenzinger Albrecht A Krijgsveld Jeroen J

Molecular systems biology 20200101 1

High-throughput and streamlined workflows are essential in clinical proteomics for standardized processing of samples from a variety of sources, including fresh-frozen tissue, FFPE tissue, or blood. To reach this goal, we have implemented single-pot solid-phase-enhanced sample preparation (SP3) on a liquid handling robot for automated processing (autoSP3) of tissue lysates in a 96-well format. AutoSP3 performs unbiased protein purification and digestion, and delivers peptides that can be directl ...[more]

PMID: 32129943

Similar Datasets

Project description:We have established a certification system for antibodies to be used in chromatin immunoprecipitation assays coupled to massive parallel sequencing (ChIP-seq). This certification comprises a standardized ChIP procedure and the attribution of a numerical quality control indicator (QCi) to biological replicate experiments. The QCi computation is based on a universally applicable quality assessment that quantitates the global deviation of randomly sampled subsets of ChIP-seq dataset with the original genome-aligned sequence reads. Comparison with a QCi database for >28,000 ChIP-seq assays were used to attribute quality grades (ranging from âAAAâ to âDDDâ) to a given dataset. In the present report we used the numerical QC system to assess the factors influencing the quality of ChIP-seq assays, including the nature of the target, the sequencing depth and the commercial source of the antibody. We have used this approach specifically to certify mono and polyclonal antibodies obtained from Active Motif directed against the histone modification marks H3K4me3, H3K27ac and H3K9ac for ChIP-seq. The antibodies received the grades AAA to BBA (www.ngs-qc.org). We propose to attribute such quantitative grading of all antibodies attributed with the label âChIP-seq gradeâ. The NGS-QC team has setup a certification procedure for Antibodies dedicated to ChIP-seq applications based on (1)a highly reproducible pipeline for performing ChIP-seq assays including the use of an automated liquid handling platform (TECAN EVO75) for ChIP and sequencing library preparation steps and (2) the use of the NGS-QC Generator tool for providing a ChIP-seq quality grade and to evaluate other parameters like the Optimal sequencing depth, the degree of reproducibility among biological replicates and the comparison of the quality grades of the certified antibody with datasets retrieved in the public domain. This quality certification structure makes possible to assign a measured Quality Stamp to ChIP-seq grade Antibodies.

Dataset Information

Automated sample preparation with SP3 for low-input clinical proteomics

Publications

Automated sample preparation with SP3 for low-input clinical proteomics.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure