High-Performance Proteomics Using Nano‑, Capillary‑, and Microflow Chromatographic Separations

Current applications
of mass-spectrometry-based proteomics range
from single-cell to body fluid analysis, each presenting very different
demands regarding sensitivity or sample throughput. Additionally,
the vast molecular complexity of proteomes and the massive dynamic
range of protein concentrations in these biological systems require
highly performant chromatographic separations in tandem with the high
speed and sensitivity afforded by modern mass spectrometers. In this
study, we focused on the chromatographic aspect and, more specifically,
systematically evaluated proteome analysis performance across a wide
range of chromatographic flow rates (0.3–50 μL/min) and
associated column diameters using a Vanquish Neo HPLC coupled online
to a Q Exactive HF-X mass spectrometer. Serial dilutions of HeLa cell
line digests were used for benchmarking, and the total analysis time
from injection to injection was intentionally fixed at 60 min (24
samples per day). The three key messages of the study are that (i)
all chromatographic flow rates are suitable for high-quality proteome
analysis, (ii) capLC (1.5 μL/min) is a very robust, sensitive,
and quantitative alternative to nLC for many applications, and (iii)
showcased proteome, phosphoproteome, and drug proteome data provide
sound empirical guidance for laboratories in selecting appropriate
chromatographic flow rates and column diameters for their specific
applications.