Automated sample preparation workflow for tandem mass tag-based proteomics

ABSTRACT: Although tandem mass tag (TMT)-based isobaric labeling has become a powerful technique for multiplexed protein quantitation, it has not been easy to automate the workflow for widespread adoption. This is because preparation of TMT labeled peptide samples involves multiple steps ranging from protein extraction, denaturation, reduction and alkylation to tryptic digestion, desalting, labeling with TMT reagents and cleanup, all of which require a high level of proficiency. The variability resulting from multiple processing steps is inherently problematic especially with large-scale studies such as clinical studies that involve hundreds of samples where reproducibility is critical for quantitation. Here, we sought to compare the performance of a recently introduced platform, AccelerOme, for automated proteomics workflows for TMT-labeling experiments with manual processing of samples. Cell pellets were prepared and subjected to a 16-plex experiment using the automated platform and a conventional manual protocol. Single shot LC-MS/MS analysis revealed a higher number of proteins and peptides identified using the automated platform. Efficiencies of tryptic digestion, alkylation and TMT labeling were similar both in manual and automated process. In addition, comparison of quantitation accuracy and precision showed similar performance in automated workflow compared to manual sample preparation. Overall, we demonstrated that the automated platform performs at a level similar to manual process in TMT-based proteomics. We expect that the automated workflow will increasingly replace manual work and be applied to large-scale TMT-baed studies providing robust results and high sample throughput.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Akhilesh Pandey  

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD040788 | Pride | 2023-10-24

REPOSITORIES: Pride