Czech Society for Mass Spectrometry

(13^th Czech Mass Spectrometry Conference and 11^th Informal Proteomic Meeting - WeO-02)
Optimization of microflow LC-MS method by design of experiments and its application to proteomics analysis

Jiří Urban ^1 *, Jan Valášek ¹, Antonín Bednařík ¹, Martina Nechvátalová ¹, Jan Preisler ¹

1. Masaryk University, Faculty of Science

Abstract

Due to the reduced radial dilution of analytes, liquid chromatography-mass spectrometry (LC-MS) utilizing narrow columns with an internal diameter of 50 – 75 μm is almost exclusively used in ultra-trace proteomics analysis of limited sample amounts. Micro-flow LC-MS employing columns with an inner diameter of 1 mm is a viable alternative in cases where the sample amount is not limited. It offers robust qualitative and quantitative performance characteristics, simplicity of implementation, and an extensive range of high-quality micro-flow columns. Additionally, micro-column LC analysis is readily applicable to routine clinical applications.
In this work, we have employed the design of experiments protocol to optimize the micro-flow LC-MS method and to investigate the primary factors controlling its sensitivity in bottom-up proteomics analysis.
We divided the optimization process into several consecutive steps planned by Box-Behnken designs. Signal intensity and the number of identified peptides were all used as optimization criteria. In liquid chromatography, we tested the effect of mobile phase flow rate, column temperature, and gradient time. In electrospray coupling, we optimized the position of the capillary, its temperature, voltage, and the flow of sheath, auxiliary, and sweep gases. Finally, we focused on optimizing the ion injection time, automatic gain control target, and Orbitrap analyzer resolution utilizing full MS and MS/MS scans.
During the optimization process, the signal intensity and number of identified peptides increased by a factor of four and two and a half times, respectively. After optimizing with a semi-complex proteomic sample, we analyzed the real-life sample and compared the obtained results with those in the literature.

* Corresponding author: urban@chemi.muni.cz

Acknowledgement:

Financial support by the Czech Science Foundation project 23-07581S is gratefully acknowledged.