An Enzyme Assisted RP-RPLC Approach for In-Depth Human Liver Phosphoproteome Analysis — ASN Events

An Enzyme Assisted RP-RPLC Approach for In-Depth Human Liver Phosphoproteome Analysis (#125)

Yangyang Bian 1 , Chunxia Song 1 , Kai Cheng 1 , Mingming Dong 1 , Fangjun Wang 1 , Junfeng Huang 1 , Deguang Sun 2 , Liming Wang 2 , Mingliang Ye 1 , Hanfa Zou 1
  1. Dalian Institute of Chemical Physics, Dalian, China
  2. The Second Affiliated Hospital of Dalian Medical University, Dalian, China

  The multidimensional separations were essential for phosphoproteomics analysis, and the introduction of protease other than trypsin had been successfully used to increase phosphoproteomics coverage. Here an enzyme assisted RP-RPLC approach, combining the multi-enzyme digestion and multi-separation, was developed for peptides separation. The Glu-C generated peptides were first offline fractionated by C18 RPLC, then each fraction was digested with trypsin. As the cleavage specificity was highly complementary for Glu-C and trypsin, the hydrophobicity of most of these peptides would change after trypsin digestion. Therefore, the peptides in each fraction showed highly orthogonal separation in the second dimensional C18 RPLC. This approach was further used for in-depth analysis of human liver phosphoproteome by combining the two mass spectrometers, Triple TOF 5600 and Orbitrap Velos. A total number of 22446 phosphorylation sites were identified from human liver tissue, corresponding to 6526 nonredundant phosphoproteins. To the best of our knowledge, this is the largest phosphoproteome dataset of human liver up to now.