Evaluating Chromatographic Approaches for the Quantitative Analysis of a Human Proteome on Orbitrap-Based Mass Spectrometry Systems

The Orbitrap is now a core component
of several different instruments.
However, evaluating the capabilities of each system is lacking in
the field. Here, we compared the performance of multidimensional protein
identification (MudPIT) on Velos Pro Orbitrap and Velos Orbitrap Elite
mass spectrometers to reversed phase liquid chromatography (RPLC)
on a Q-Exactive Plus and an Orbitrap Fusion Lumos. Using HeLa cell
protein digests, we carried out triplicate analyses of 16 different
chromatography conditions on four different instrumentation platforms.
We first optimized RPLC conditions by varying column lengths, inner
diameters, and particle sizes. We found that smaller particle sizes
improve results but only with smaller inner diameter microcapillary
columns. We then selected one chromatography condition on each system
and varied gradient lengths. We used distributed normalized spectral
abundance factor (dNSAF) values to determine quantitative reproducibility.
With Pearson product-moment correlation coefficient r values routinely above 0.96, single RPLC on both the QE+ and Orbitrap
Lumos outperformed MudPIT on the Orbitrap Elite mass spectrometer.
In addition, when comparing dNSAF values measured for the same proteins
across the different platforms, RPLC on the Orbitrap Lumos had greater
sensitivity than MudPIT, as demonstrated by the detection and quantification
of histone deacetylase complex components. Data are available via
ProteomeXchange with identifier 10.6019/PXD009875.