Repurposed 3D printer allows economical and programmable fraction collection for proteomics of nanogram scale samples

Introduction

Two-dimensional liquid chromatography (2DLC) is a powerful tool for the analysis of complex proteomes. However, it suffers from sample losses during transfer between separation steps, compromising sensitivity and quantitative accuracy in LC-MS analysis, restricting the use of 2DLC to studies where abundant material is readily available. For studies involving samples with limited availability, achieving adequate analytical depth while minimizing sample losses presents a significant challenge. To address this issue, in this work, we built and applied a low loss fractionation system comprised of a reversibly reconfigured Evosep One LC system for the first dimension and a repurposed 3D-printer as a fraction collector in a range of Proteomics workflows covering both data dependent and data independent approaches as well as phosphoproteomics experiment.

Methods

Peptides from human cell lysates were analysed by 2D RP-RP. An Evosep One LC system was used for the first-dimension separation in combination with a modified 3D printer as a fraction collector. Fractionated and non-fractionated samples were analysed by low-pH LC-MS/MS on an Ultimate 3000 RSLCnano system coupled to an Orbitrap Ascend Tribrid mass spectrometer (Thermo Fisher Scientific) using Data Dependent (DDA), Data Independent (DIA) or Wide Window Acquisition (WWA) modes. The resulting MS raw data were analysed by FragPipe, MaxQuant, DIA-NN or Proteome Discoverer. Data analysis was performed using the tools available in Perseus and GraphpadPrism.

Results

The 2D RP-RP system used in this work demonstrated superior proteome coverage over single-shot experiments, achieving high sensitivity at peptide level for low amount of input material. More importantly, increase in protein level intensity was observed in 2D RP-RP, which indicates the complexity reduction afforded by the system outweighs the sample losses endured. The application of DIA and WWA to fractionated samples allowed further improvements in peptide and protein IDs in comparison to DDA, demonstrating that the use of data independent acquisition methods also benefit from the complexity reduction caused by fractionation. The utility of the 2D system was further investigated in phosphoproteomics workflow, showing significant improvements in phosphosites and phosphoproteins over single-shot analysis.

Conclusions

Our 2D workflow showed significant increases in peak capacity and identification rates over single-shot experiments in the analysis of human cell lysates, exhibiting superior proteome coverage using limited sample amounts. Moreover, substantial improvements in coverage were also observed when the workflow was applied to the analysis of subproteomes. We envision that the 2D system showed here will have broad applicability across various proteomics workflows and could be particularly useful for isobaric multiplexed experiments applied to small cell populations.