Poster Presentation 28th Annual Lorne Proteomics Symposium 2023

Quantitative Multiplexed Analysis of Cancer Cell Lines Using Automated Sample Preparation with Increased Speed and Sensitivity (#168)

Woong Kim 1 , Steven L. Reeber 1 , Frank Rooney 2 , Vlad Zabrouskov 1 , Daniel Lopez Ferrer 1
  1. Thermo Fisher Scientific, San Jose, CA, USA
  2. Thermo Fisher Scientific, Scoresby, VIC, Australia

 

Immortalized cell lines have played a key role in understanding the underlying biology underlying and as screening tools to identify drug targets in different signaling pathways related to diseases. Highly sensitive analytical method combined with standardized method for high quality samples preparation are essential in quantitative proteomics. Here we describe an end to end solution for quantitative proteomics analysis including automated sample preparation with customized reagent kit and Orbitrap Ascend using TMTproTM workflow.

Sixteen different colon cancer cell lines were grown in four different batches and harvested in four different dates to assess the variability of a normal multiplexed experiment. 1x106 cells from each cell line was harvested and rinsed with PBS. All the samples were processed using AccelerOme TMTproTM kit was used together with the AccelerOme system. Peptides were analyzed on Orbitrap Ascend Tribrid Mass Spectrometer coupled to VanquishTM Neo UHPLC system. Acquired data was analyzed using Proteome DiscovererTM software.

In quantitative analysis of large numbers of samples, quality of sample preparation, high confidence analysis with high throughput analysis are key aspects to address. AccelerOme system resulted in digestion efficiency above 92% and TMT labeling efficiency above 99% as the reaction chemistry while keeping artifactual modification below 1%. More importantly, sample preparation time were decreased from three days to six hands free hour, reducing the sample preparation time to 30% compared to a traditional manual method. The samples were analyzed using real time search (RTS) in Orbitrap Ascend Tribrid where architecture improvement has increased the ion management resulting in higher speed and sensitivity of peptide analysis and resulted in increase of 20% more peptides being quantified.

AccelerOme together with Ascend provides an ideal accurate and precise multiplexed proteomics workflow for mammalian cell line based proteomics research.