The use of LysargiNase complementary to trypsin in large scale phosphoproteome study for unambiguous phosphosite localization

ABSTRACT: Understanding of kinase-guided signaling pathways requires identification and analysis of phosphorylation sites. Mass spectrometry (MS)-based phosphoproteomics is a rapid and highly sensitive approach for high-throughput identification of phosphorylation sites. However, the exact localization of phosphorylation sites which depends on trypsin-centric shotgun phosphoproteomics is often arbitrary and unreliable because of the limited product ion coverage in the MS2 spectra. Therefore, phosphorylation site determination from MS data with a single protease cannot be expected to be comprehensive, regardless of the strategy used for phosphopeptide detection. Here, we have explored the application of LysargiNase which was recently reported to mirror trypsin in specificity to cleave arginine and lysine residues exclusively at the N-terminal side. Our data demonstrates that the combined application of LysargiNase and trypsin results in higher sequence coverage with more complete ladder sequences which is helpful to pinpoint the precise phosphorylation sites. Therefore, the combined use of these two enzymes will partially overcome the limitations of trypsin-centric phosphoproteomics to achieve a more reliable phosphoproteome and to increase the identification of novel phosphorylation sites.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hepatocyte, Liver

SUBMITTER: Feng Xu  

LAB HEAD: Ping Xu

PROVIDER: PXD011178 | Pride | 2020-05-22

REPOSITORIES: Pride