LysargiNase enhances protein identification on the basis of trypsin on formalin-fixed paraffin-embedded samples

Abstract

Rationale: Formalin-Fixed Paraffin-Embedded (FFPE) samples are valuable for proteomic studies of disease. However, the crosslink among proteins, protein vs nucleic acid, and other covalent chemical modifications like methylation introduced by formaldehyde can interfere with trypsin digestion in proteomics studies. LysargiNase was reported to have a better full-cleavage rate at methylation and b ion coverage than trypsin. The contribution of LysargiNase in the proteomic study of FFPE samples was assessed and compared with trypsin in this study for the first time to facilitate proteomic research on FFPE samples.

Methods: The FFPE proteins were extracted with an "antigen retrieval" method. Digestion parameters were optimized by visualization of the digests on the tricine gel by silver staining. Then the FFPE proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and cut into 16 gel bands and in-gel digested by trypsin and LysargiNase, respectively. Peptides were desalted with Stage-Tips and separated via liquid chromatography. Electrospray ionization was conducted and peptide mass was measured in the LTQ Orbitrap Velos in the data-dependent mode.

Results: High concentrations of enzyme facilitate the digestion efficiency of FFPE samples. A total of 32,294 peptides and 3445 proteins were identified with LysargiNase and trypsin combined in two replicates. LysargiNase increased peptide identification by 18.9% and protein identification by 13.4% on the basis of trypsin. Consistently, LysargiNase increased C-terminal peptide identification by 47.7%. Moreover, LysargiNase showed better full-cleavage rate (49.3%) at methylated sites than trypsin (23.9%). LysargiNase and trypsin combined can improve the b-ion coverage by 50% on FFPE samples.

Conclusions: FFPE samples can be more efficiently digested at high concentrations of LysargiNase and trypsin. LysargiNase can better digest methylated peptides and improve the proteome identification by 13.4% and the b-ion coverage by 50% on the basis of trypsin in FFPE samples.