Throughput limitations often preclude the adoption of nanoLC-MS methods for translational proteomics applications such as biomarker validation because the large sample cohorts need to be analyzed to reveal changes that stand out of biological, analytical, and sample preparation variation.
The next-generation nano-, capillary- and micro-flow UHPLC systems were installed across three sites (San Jose, US; Germering, Germany; Bremen, Germany) and coupled with Orbitrap Exploris 480 HRAM MS. HeLa protein digest were separated on EASY-Spray columns (75 µm x 15 cm, 2 µm) with trap-and-elute workflow. The five LC-MS methods with cycle times of 8, 14.4, 24, 48, and 60 min and data-dependent MS acquisition were tested across three sites to assess the reproducibility of peptide and protein identifications and quantification precision. The data were processed Proteome Discoverer 2.5 software. Results were filtered for 1% FDR on peptide and protein levels.
Five robust standard LC-MS methods operated at flow rates in the range from 1.3 to 0.3 µL/min have been developed to maximize MS utilization (calculated as the ratio of peptide elution window to cycle time). The MS utilization gradually increased from 68% for the shortest method to 95% for the longest 60 min method. The length of the methods is also linked to the increased FWHM from ca. 3 sec for 8 min method to ca. 10 sec for 60 min method. To comprehensively estimate the analytical variability were compared the quantification and identification results collect (i) on the same low-flow LCMS instrument using the same separation column; (ii) on the same instrument using three separation columns of the same dimensions; (iii) on different instruments, with different separation and trap columns, located at three different laboratories in the US and Europe. On average we identified 1218, 2063, 2881, 3810, 4051 high confident proteins with 8, 14.4, 24, 48, and 60 min methods with < 5% RSD between separation columns. We found that more than 72.5% of proteins were commonly identified across three laboratories independent of the method length. While using 14.4 min methods for 100 injections were observed stable retention times and 2800 protein and 12K peptide identifications. The label-free quantification showed that 72% of identified proteins have less than 25% variation of abundance for 100 continuous injections. The obtained results allow setting realistic estimates for abundance variation that are required to confidently detect targets above the multi-site analytical variation.