Online Alkaline-pH Reversed-Phase Nanoelectrospray-Tandem Mass Spectrometry Complements Traditional Phosphoproteomic Analysis via Influencing Charge State Distribution of Phosphopeptides

Phosphorylation
(O-linked and N-linked) plays an
important role in biological functions and cell
signaling. Here, we employed a one-dimensional online alkaline-pH
reversed-phase nanoelectrospray-tandem mass spectrometry (alkaline-pH-MS/MS)
for the investigation of global phosphorylation. In this method, phosphopeptides
were separated on a nanoflow C18 column with an alkaline-pH gradient
and directly introduced to the mass spectrometer through nanoelectrospray
ionization. Although the phosphosites and phosphopeptides identified
by alkaline-pH-MS/MS were slightly lower than those of traditional
online low-pH reversed-phase tandem MS (low-pH-MS/MS), these two methods
were highly complementary to each other. This alkaline-pH-MS/MS may
affect the actual polarity and CSD of phosphopeptides, consequently
improving the identification of multiply phosphorylated peptides.
Moreover, alkaline-pH-MS/MS was compatible with other peptide fractionation
and phosphopeptide enrichment techniques, such as offline high-pH
or low-pH reversed-phase liquid chromatography fractionation. The
complementarity of alkaline-pH-MS/MS and low-pH-MS/MS was further
demonstrated by the tandem mass tag (TMT)-based quantitative phosphoproteomic
analysis of five pairs of hepatocellular carcinoma (HCC) tumors and
normal adjacent tissues (NATs). Furthermore, unique information on
significantly changed phosphosites was observed by alkaline-pH-MS/MS.
This study provided an alternative and complementary tool for global
analysis of both O- and N-phosphoproteome,
which may be beneficial for the discovery of phosphoproteins with
significant biological functions.