Long-term stability and reproducibility of nano-, capillary- and micro-flow LC-MS separations

Introduction
Nano-, capillary- and micro-flow LC hyphenation with MS detection is irreplaceable in a high-sensitivityanalysis of complex bio-samples. But concerns remain around long-term stability, day-to-day as well assystem-to-system results reproducibility. The transition of low-flow LC-MS applications from academic andR&D to translational research laboratories demands to prove the suitability of LC and MS technologies forthe analysis of large sample cohorts. In addition, the consistency of results across multiple laboratories andoperators is essential for pooling datasets for joint statistical analysis. Here we describe the output ofcomprehensive evaluation of proteomics data collected over several months of continuous operation atdifferent laboratories and discuss the impact of hardware and consumables on the reproducibility of results.

Methods
Low-flow LC-MS and/or LC-UV data were collected in Germering (Germany), Bremen (Germany), and SanJose (USA) by different operators using identical LC and MS hardware and different batches of separationcolumns. The Vanquish Neo UHPLC system was used for peptides separation on 75 μm ID EASY-SprayPepMap Neo columns with 15, 50, and 75 cm length and 1 mm ID columns with 15 cm length. OrbitrapExploris 480 and Orbitrap Exploris 240 MS instruments were used to detect and fragment peptides anddata-dependent acquisition mode. Data were processed with and without resolving of chimeric spectra andreported with 1% FDR on peptide and protein level.

Preliminary Data
Long-term robustness.
The continuous injections of bovine serum albumin protein digest under typical fornanoLC-MS bottom-up proteomics experimental conditions were conducted for at least 6 months in twolaboratories. The longevity results of column backpressure, retention time variation, and full-width half-max(FWHM) for selected peptide peaks showed the suitability of low-flow separations LC for the uninterruptedlong-term analysis.

Novel Aspect
A comprehensive evaluation of analytical variability in low-flow LC-MS analysis provides guidelines forachieving reproducible results across instruments and laboratories