In Search of a Universal Method: A Comparative Survey of Bottom-Up Proteomics Sample Preparation Methods

Robust, efficient, and reproducible protein extraction
and sample
processing is a key step for bottom-up proteomics analyses. While
many sample preparation protocols for mass spectrometry have been
described, selecting an appropriate method remains challenging since
some protein classes may require specialized solubilization, precipitation,
and digestion procedures. Here, we present a comprehensive comparison
of the 16 most widely used sample preparation methods, covering in-solution
digests, device-based methods, and commercially available kits. We
find a remarkably good performance of the majority of the protocols
with high reproducibility, little method dependency, and low levels
of artifact formation. However, we revealed method-dependent differences
in the recovery of specific protein features, which we summarized
in a descriptive guide matrix. Our work thereby provides a solid basis
for the selection of MS sample preparation strategies for a given
proteomics project.