Poster Presentation 29th Annual Lorne Proteomics Symposium 2024

Single-shot LC-MS workflow for comprehensive proteome identification on a novel high-resolution accurate mass platform. (#188)

Santosh Renuse 1 , Tabiwang N. N Arrey 2 , Anna Pashkova 2 , Maowei Dou 3 , Jeff Op De Beeck 4 , Ryan D Bomgarden 3 , Bernard Delanghe 2 , Eugen N Damoc 2 , Sally Webb 1 , Joanne M Ford 5
  1. Analytical Instruments Group, Thermo Fisher Scientific, San Jose, San Jose, California, USA
  2. Analytical Instruments Group, Thermo Fisher Scientific, GmbH, Bremen, Germany
  3. Life Sciences Solutions Group, Thermo Fisher Scientific, Rockford IL, Rockford, Illinois, USA
  4. Analytical Instruments Group, Thermo Fisher Scientific, Belgium, Ghent, Belgium
  5. Thermo Fisher Scientific, Scoresby, VIC, Australia

Recent reports on single-shot proteomic analyses showed identification of up to 10,000 proteins at 6-10 samples per day throughput. Such methods are not suitable for analysis of medium to large sample cohorts considerably adding sample to sample variation. Ultra-long gradient methods also have issues in consistent protein quantitation due to variation in peptide retention times and low throughput. Here, we present a single-shot LC-MS/MS 30 SPD workflow with identification of >10,000 protein groups from human cell lines on the new Orbitrap Astral platform.

HeLa, MCF7, HCT116, A549 and CHO cell line lysates were processed using the AccelerOme automated sample preparation platform for reduction, alkylation, in-solution trypsin digestion and peptide clean-up. The digested peptides were vacuum dried and reconstituted in 0.1% FA. The reconstituted peptides (5 ng, 10 ng, 50 ng, 100 ng, 200 ng and 500 ng) were separated using a mPAC™ column (50 cm with pillar diameter of 180 mm and interpillar distance of 16 mm) on the Orbitrap Astral mass spectrometer interfaced with the Vanquish Neo™ UHPLC system. The raw files were analyzed using CHIMERYS in Proteome Discoverer 3.1.

Single-shot analyses also have advantages for handling large cohort samples with different sample types such as cells, tissues, and body fluids. Efforts have been undertaken to get in-depth proteome coverage in single shot analyses. However, it often involves ultra-long LC gradients which hampers sample throughput. Here, we used a single-shot DIA LC-MS workflow to identify proteins in several cell lines. The workflow described here consists of the Vanquish Neo UHPLC system, mPAC™ Neo 50cm column, EASY-Spray™ ion source, the Orbitrap Astral mass spectrometer and a new version of Proteome Discoverer software suite. Using this workflow, we identified 9,305, 9,456, 10,363, 9,783 and 9,395 protein groups from HeLa, MCF7, HCT116, A549 and CHO cell lines, respectively with 500 ng peptide loads on mPAC™ Neo 50cm column. Of these identifications, 70-80% of identified proteins had CV < 20%. The average unique peptides identifications from these cell lines were in the range of 70,000 to 105,000. We tested the single-shot LC-MS workflow at different sample loads from 5 ng to 500 ng and identified 5,000 -10,000 protein groups. The unique peptides identifications at these loads were from 40,000 to 100,000. This clearly demonstrates the utility of our single-shot LC-MS workflow for in-depth proteome analysis. Results from higher-throughput methods (i.e., 60, 100 and 180SPD) will also be presented.