iProXProteomicsHu ZhouHomo Sapienshttp://www.iprox.org/page/project.html?id=IPX0004181000zhouhu@simm.ac.cnShanghai Institute of Materia MedicaiProXThe 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 (p<i>I</i>). 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.Online Alkaline-pH Reverse Phase Nanoelectrospray-Tandem Mass Spectrometry Complements Conventional Low-pH Method for Global Proteomic Analysis.Gao Jing J, Wang Yuqiu Y, Zhang Shu S, Yang Zhicheng Z, Zhu Hongwen H, Luo Xuanyang X, Lu Dayun D, Gao Qiang Q, Zhou Hu HfalseOnline alkaline-pH reverse phase nanoelectrospray-tandem mass spectrometry complements conventional low-pH method for global proteomic analysisThe 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 (AlkalinepH-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) and directly introduced to the mass spectrometer through nanoelectrospray ionization. The identified peptides overlapped between AlkalinepH-MS/MS and conventional online low-pH reverse phase nanoelectrospray-tandem mass spectrometry (LowpH-MS/MS) were as low as 45%, indicating that these two methods were complementary to each other. In addition, the AlkalinepH-MS/MS method showed identification capacity for a higher proportion of peptides with low molecular weight (MW), positive grand average of hydropathy (GRAVY), or high isoelectric point (pI). Moreover, the AlkalinepH-MS/MS method had a bias toward histidine, lysine, methionine, and proline- containing peptides. The complementarity of AlkalinepH-MS/MS and LowpH-MS/MS was further demonstrated for the analysis of tryptic digests from 15 intrahepatic cholangiocarcinoma (iCCA) cell lines. The alternating of 60-min AlkalinepH-MS/MS and 60-min LowpH-MS/MS method outperformed the conventional 120-min LowpH-MS/MS method in the identification of amino acid variants and protein groups. Our AlkalinepH-MS/MS method was an excellent complementary and alternative method to the LowpH-MS/MS method for global proteomic analysis.Sun Jan 15 00:00:00 GMT 2023PXD039453960636751022