Repurposed 3D Printer Allows Economical and Programmable Fraction Collection for Proteomics of Nanogram Scale Samples

In this work, we describe the construction and application
of a
repurposed 3D-printer as a fraction collector. We utilize a nano-LC
to ensure minimal volumes and surfaces although any LC can be coupled.
The setup operates as a high-pH fractionation system capable of effectively
working with nanogram scales of lysate digests. The 2D RP–RP
system demonstrated superior proteome coverage over single-shot data-dependent
acquisition (DDA) analysis using only 5 ng of human cell lysate digest
with performance increasing with increasing amounts of material. We
found that the fractionation system allowed over 60% signal recovery
at the peptide level and, more importantly, we observed improved protein
level intensity coverage, which indicates the complexity reduction
afforded by the system outweighs the sample losses endured. The application
of data-independent acquisition (DIA) and wide window acquisition
(WWA) to fractionated samples allowed nearly 8000 proteins to be identified
from 50 ng of the material. The utility of the 2D system was further
investigated for phosphoproteomics (>21 000 phosphosites
from
50 μg starting material) and pull-down type experiments and
showed substantial improvements over single-shot experiments. We show
that the 2D RP–RP system is a highly versatile and powerful
tool for many proteomics workflows.