Increased Sensitivity of Mass Spectrometry by Alkaline Two-Dimensional Liquid Chromatography: Deep Cover of the Human Proteome in Gene-Centric Mode

Currently, great
interest is paid to the identification of “missing”
proteins that have not been detected in any biological material at
the protein level (PE1). In this paper, using the Universal Proteomic
Standard sets 1 and 2 (UPS1 and UPS2, respectively) as an example,
we characterized mass spectrometric approaches from the point of view
of sensitivity (Sn), specificity (Sp), and accuracy (Ac). The aim
of the paper was to show the utility of a mass spectra approach for
protein detection. This sets consists of 48 high-purity human proteins
without single aminoacid polymorphism (SAP) or post translational
modification (PTM). The UPS1 set consists of the same 48 proteins
at 5 pmols each, and in UPS2, proteins were grouped into 5 groups
in accordance with their molar concentration, ranging from 10–11 to 10–6 M. Single peptides from
the 92% and 96% of all sets of proteins could be detected in a pure
solution of UPS2 and UPS1, respectively, by selected reaction monitoring
with stable isotope-labeled standards (SRM-SIS). We also found that,
in the presence of a biological matrix such as Escherichia
coli extract or human blood plasma (HBP), SRM-SIS makes it
possible to detect from 63% to 79% of proteins in the UPS2 set (sensitivity)
with the highest specificity (∼100%) and an accuracy of 80%
by increasing the sensitivity of shotgun and selected reaction monitoring
combined with a stable-isotope-labeled peptide standard (SRM-SIS technology)
by fractionating samples using reverse-phase liquid chromatography
under alkaline conditions (2D-LC_alk). It is shown that this technique
of sample fractionation allows the SRM-SIS to detect 98% of the single
peptides from the proteins present in the pure solution of UPS2 (47
out of 48 proteins). When the extracts of E. coli or Pichia
pastoris are added as biological matrixes to the UPS2, 46,
and 45 out of 48 proteins (∼95%) can be detected, respectively,
using the SRM-SIS combined with 2D-LC_alk. The combination of the
2D-LC_alk SRM-SIS and shotgun technologies allows us to increase the
sensitivity up to 100% in the case of the proteins of the UPS2 set.
The usage of that technology can be a solution for identifying the
so-called “missing” proteins and, eventually, creating
the deep proteome of a particular chromosome of tissue or organs.
Experimental data have been deposited in the PeptideAtlas SRM Experiment
Library with the dataset identifier PASS01192 and the PRIDE repository
with the dataset identifier PXD007643.