Reports of analysis of the human liver phosphoproteome analysis in the literature has been very limited.Here,an enzyme assisted RP-RPLC approach was developed,and we demonstrate that the introduction of trypsin digestion for fractionated Glu-C generated peptides led to a wider separation window at the second RPLC.The approach was further combined with two different types of mass spectrometers,TripleTOF(R) 5600 system and LTQ Orbitrap Velos for the mapping of human liver phosphoproteome.TripleTOF(R) 5600 system and LTQ Orbitrap Velos resulted in 13976 and 14050 phosphorylation sites separately,resulting in a total number of 22446 modification sites identified,corresponding to 6526 nonredundant phosphoproteins from human liver tissue.It is interested that only 25% of the total phosphorylation sites were both identified by the two instruments,this clearly demonstrated that the coverage of phosphorylation sites could be significantly increased with the using different type of mass spectrometers.So far this is the largest data set of phosphoproteome of human liver up to now.