Dataset Information

TECAN Resolvex A200 Positive Pressure Workstation for Semi Automated Peptide Purification of Complex Proteomic Samples

ABSTRACT: High-throughput has become a substantial need in many omics investigations. In proteomics the sample preparation workflow consists of multiple steps, whereby with each additional manual step more bias is added to the sample. Especially for label-free quantification experiments this drastically reduces the outcome. Here, a positive pressure workstation was evaluated to increase automation of sample preparation and decrease input time and consumables. The semi-automated workflow with the Resolvex A200 workstation including the sorbents Maestro and WWP2 performed equally well to the routinely conducted manual workflow, with similar technical variability in MSMS-based identifications of peptides and proteins. Both sorbents produced highly reproducible results within a range of 10-300 µg of peptide starting material. A direct, on-column sample preparation using the NBE columns - without conditioning and equilibration of the sorbents -works generally robust, whereby consumables and time are drastically reduced.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Cricetulus Griseus (chinese Hamster) (cricetulus Barabensis Griseus)

TISSUE(S): Cell Suspension Culture, Permanent Cell Line Cell

SUBMITTER: Louise Schelletter

LAB HEAD: Thomas Noll

PROVIDER: PXD018444 | Pride | 2021-01-25

REPOSITORIES: Pride

ACCESS DATA

Publications

A positive pressure workstation for semi-automated peptide purification of complex proteomic samples.

Schelletter Louise L Hertel Oliver O Antar Shareef Jarvi SJ Scherling Christian C Lättig Jens J Noll Thomas T Hoffrogge Raimund R

Rapid communications in mass spectrometry : RCM 20200817 2

<h4>Rationale</h4>High-throughput reliable data generation has become a substantial requirement in many "omics" investigations. In proteomics the sample preparation workflow consists of multiple steps adding more bias to the sample with each additional manual step. Especially for label-free quantification experiments, this drastically impedes reproducible quantification of proteins in replicates. Here, a positive pressure workstation was evaluated to increase automation of sample preparation and ...[more]

PMID: 32583429

Similar Datasets

Project description:The analysis of single cell proteomes has recently become a viable complement to transcript and genomics studies. Proteins are the main driver of cellular functionality and mRNA levels are often an unreliable proxy of such. Therefore, the global analysis of the proteome is essential to study cellular identities. Both multiplexed and label-free mass spectrometry-based approaches with single cell resolution have lately attributed surprising heterogeneity to believed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lacks behind. Here, we introduce the proteoCHIP, a universal option for single cell proteomics sample preparation at surprising sensitivity and throughput. The automated processing using a commercial system combining single cell isolation and picoliter dispensing, the cellenONE®, allows to reduce final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error prone manual sample handling and overcoming evaporation. With this specialized workflow we achieved around 1,000 protein groups per analytical run at remarkable reporter ion signal to noise while reducing or eliminating the carrier proteome. We identified close to 2,000 protein groups across 158 multiplexed single cells from two highly similar human cell types and clustered them based on their proteome. In-depth investigation of regulated proteins readily identified one of the main drivers for tumorigenicity in this cell type. Our workflow is compatible with all labeling reagents, can be easily adapted to custom workflows and is a viable option for label-free sample preparation. The specialized proteoCHIP design allows for the direct injection of label-free single cells via a standard autosampler resulting in the recovery of 30% more protein groups compared to samples transferred to PEG coated vials. We therefore are confident that our versatile, sensitive, and automated sample preparation workflow will be easily adoptable by non-specialized groups and will drive biological applications of single cell proteomics.

Dataset Information

TECAN Resolvex A200 Positive Pressure Workstation for Semi Automated Peptide Purification of Complex Proteomic Samples

Publications

A positive pressure workstation for semi-automated peptide purification of complex proteomic samples.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure