Standardization of technology platforms and methods is a key for wide adoption of LCMS in different applied areas. However, in reality, the number of different instruments, setups, configurations, and methods for typical LCMS proteome profiling is extremely high that complicates the transfer of the developed method.
The typical proteomics workflow includes experiments for deep dive proteome analysis followed by an analysis of large sample cohorts with high-throughput, sensitive and robust LCMS. Whilst nanoLCMS is capable of both specific and significant proteome depth, its widespread adoption has been limited by the low throughput and insufficient robustness of the methods employed.
Here we demonstrate a new set of capillary-flow LC-MS (capLC-MS) methods capable of large sample cohort analysis using a Thermo Scientific™ UltiMate™ 3000 RSLCnano system coupled to a new Thermo Scientific™ Exploris™ 480 Hybrid Quadrupole-Orbitrap™ mass spectrometer.
The five low-flow LC-MS methods are capable of throughputs of 180, 100, 60, 30 and 24 samples per day affording MS utilization from 75 to over 90% respectively. These were validated using HeLa protein and crude plasma digests and showed excellent long term reproducibility and good protein coverage with more than 150 protein groups identified using the 8 min LC-MS method increasing to over 250 protein groups for the 60 min LC-MS plasma runs. The results were compared with standard nanoLCMS with 45 and 90 min peptide elution window.
The low-flow LCMS methods in combination with the high sensitivity and fast acquisition speed of the Exploris 480 mass spectrometer provide robust, fast and quantitative and deep profiling for complex sample matrices including crude plasma protein digests and cell lysates. Furthermore, the consistent results generated over hundreds of replicate injections, prove that the methods are suited to the analysis of large sample cohorts and biomarker studies.