Proteomic analyses of large cohorts of human patient samples plays a critical role in discovering novel biomarkers for diagnosis of disease and responses to treatment. Here, we introduce a novel packed emitter chromatography column, the IonOpticks Aurora Rapid 8x150 (8 cm x 150 μm inner diameter, 1.7 μm C18), designed to improve the performance of large cohort proteomic analyses. Quality control samples embedded within a large human patient cohort were used to assess the efficiency and reproducibility of the column.
A HeLa tryptic digest (200 ng) was separated on Aurora Rapid 8 cm x 150 μm column on a 60SPD method using Evosep One and Thermo Fisher Orbitrap Astral Mass Spectrometer. MS parameters: MS1 m/z range(Orbi)/MS2(Astral) from 380 to 980, isolation windows: 2Th, maximum injection time of 3 ms, FAIMS compensation voltage (CV): -40, resolution for MS1 set at 120,000. Patient plasma samples were analysed using 100 samples per day method with approximately 700 samples run between quality control (QC) injections. QC injections were included to monitor system performance (acquired using 60 sampler per day method).The data were processed using DIA-NN (version 1.8.1) with match between runs enabled.
The IonOpticks Aurora Rapid 8x150, paired to the EvoSep One and Thermo Fisher Orbitrap Astral Mass spectrometer achieved robust protein identifications, with over 9,300 proteins and 100,000 unique peptides identified per QC run. Coefficient of variation, Pearson correlation and average peak width were calculated to assess column reproducibility across the cohort. A median CV of 2.52 %, >0.98 Pearson correlation score and 2.9 second FWHM were achieved across the 257 QC analyses.
Our data demonstrates that the novel packed emitter column combined with the Evosep One and Thermo Fisher Orbitrap Astral Mass Spectrometer enabled maximum identifications whilst ensuring robust and reproducible protein quantification across a large patient plasma cohort analysis.