on 09-20-201902:34 PM - edited on 10-15-202111:30 AM by Closed Account
Oleksandr Boychenko1, Christopher Pynn1, Mike Baynham2, Jenny Ho3, Tabiwang Arrey4, and Peter Jehle1 Hupo 2019 Purpose: The availability and ease of sampling of blood and its constituents make it ideal for large cohort clinical research studies. Proteins, as major players in catalytic and structural as well as many signaling functions, are essential research targets. 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.
Methods: Here we demonstrate novel capillary-flow LC-MS (capLC-MS) methods capable of large sample cohort analysis using a Thermo Scientific™ UltiMate™ 3000 RSLCnano system coupled to a Thermo Scientific™ Exploris™ 480 and Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap™ mass spectrometer.
Results: 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 plasma runs. The low-flow LC-MS methods provide sharp, symmetric peaks that, in combination with the high sensitivity and fast acquisition speed of the state-of the-art Orbitrap mass spectrometers, deliver robust, fast and deep profiling for 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.