Relative quantification of proteins in complex samples raises a demand for high sensitivity and reproducibility throughout large sample sets to gain meaningful insights on biological processes. Data-independent analysis has emerged as a powerful technique enabling quantification of thousands of proteins because it avoids the intensity bias and missing value problem that typically limit data-dependent methods. DIA in principle interrogates all peptides that are present in a sample and therefore is especially suitable for high-throughput and large-cohort studies.
Different microbial proteomes were spiked into a human proteome background at different ratios, yielding two proteome and three proteome mixtures with varying total protein amounts. Samples were separated on a 50 cm µPAC™ HPLC columns in direct injection setup on a Vanquish™ Neo system under nano-flow conditions. DIA experiments were run on an Orbitrap Exploris™ 240 mass spectrometer. Data was analyzed by Spectronaut™ 16 using a library-free approach.
Using micropillar array-based column technology under nano-flow conditions for separation of peptides gives optimal peak shapes and intensities reproducibly over a long-term acquired data set with minimal performance loss. In connection with the high resolution DIA methodology, this enables for wide proteome coverage in two- and three-proteome mixtures as well as quantification accuracy below 10 % at high sample throughput. The uncomplicated and easily implemented library-free data analysis yields similar performance as low-key library-based approaches.
Micropillar array-based separation technology and high-resolution data-independent analysis enable for a wide proteome coverage at high throughput, while maintaining excellent quantification accuracy of relative protein ratios in complex cell lysates.