Online Alkaline-pH Reverse Phase Nanoelectrospray-Tandem Mass Spectrometry Complements Conventional Low-pH Method for Global Proteomic Analysis

The global proteome analysis was limited by the identification
of peptides with low abundance or specific physiochemical properties.
Here, a one-dimensional online alkaline-pH reverse phase nanoelectrospray-tandem
mass spectrometry (alkaline-pH-MS/MS) method was developed and optimized
for global proteomic analysis. In this method, peptides were separated
on a nanoflow C18 column with an alkaline-pH mobile phase (pH = 8.0)
and directly injected into the mass spectrometer. The unique peptides
overlapped between alkaline-pH-MS/MS and conventional online low-pH
reverse phase nanoelectrospray-tandem mass spectrometry (low-pH-MS/MS)
were as low as 45%, strongly indicating that these two methods were
complementary to each other. In addition, alkaline-pH-MS/MS showed
identification capacity for a higher proportion of peptides with negative
grand average of hydropathy (GRAVY) or high isoelectric point (pI). Compared to low-pH-MS/MS, alkaline-pH-MS/MS enabled
enrichment preference toward histidine-, lysine-, methionine-, and
proline-containing peptides. The complementarity of alkaline-pH-MS/MS
and low-pH-MS/MS was further demonstrated for the analysis of tryptic
digests from 15 intrahepatic cholangiocarcinoma (iCCA) cell lines.
The alternating 60 min alkaline-pH-MS/MS plus 60 min low-pH-MS/MS
method outperformed the conventional 120 min low-pH-MS/MS method in
both the identification of amino acid variants and protein groups.
Therefore, we established the alkaline-pH-MS/MS method as a simple,
competitive, alternative method to low-pH-MS/MS for global proteomic
analysis.