Enhancing Sensitivity of Microflow-Based Bottom-Up Proteomics through Postcolumn Solvent Addition

The introduction of more sensitive
mass spectrometers allows researchers
to adapt front-end liquid chromatography (LC) to individual needs
for the analysis of complex proteomes. Where absolute sensitivity
is not paramount, it is advantageous to switch from a highly sensitive
nanoflow-LC setup, the de facto standard platform in mass-spectrometry
(MS)-based discovery proteomics, to a more robust, high-throughput-compatible
microflow or conventional-flow setup. To enhance the microflow-LC-MS
electrospray process of complex proteomic samples, we tested the effects
of different solvents, including 2-propanol, methanol, and acetonitrile,
pure or as mixture with dimethyl sulfoxide, which were added postcolumn
to the eluting sample. Postcolumn addition of organic solvents strongly
enhanced the electrospray efficiency in microflow-LC-MS experiments
and improved the sensitivity across the entire gradient and for early
eluting peptides by up to 10-fold. Postcolumn solvent addition did
not negatively affect chromatographic performance and resulted in
an overall 28–36% increase in identifications at both the protein
and peptide levels. The presented microflow-LC-MS workflow, including
postcolumn solvent addition, can be easily adopted on any LC-MS/MS
platform.