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.

Unambiguous Phosphosite Localization through the Combination of Trypsin and LysargiNase Mirror Spectra in a Large-Scale Phosphoproteome Study

“…In this study, we obtained about 42.5% shared peptides (Figure F), which makes it more feasible to exactly pinpoint phosphosite by combining mirror spectra. The higher number of identified peptides from our LysargiNase was likely due to our self-developed LysargiNase with higher protease activity and stability …”

“…The higher number of identified peptides from our LysargiNase was likely due to our self-developed LysargiNase with higher protease activity and stability. 25 The combined spectrum is a pseudo-spectrum generated from mirror spectra between trypsin and LysargiNase. Note that according to our definition of mirror spectra (Supporting Information Figure 2A), the spectra of different phosphosites are not mirror spectra and will not be combined.…”

ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates

“…In addition, trypsin is typically used in bottom-up proteomic studies. However, arginine methylation, especially dimethylation, affects the cleavage efficiency of trypsin and subsequently increases miscleavage rate. , The combined effect of methylarginine clustering in protein sequences and miscleavages by trypsin can potentially lead to peptides too long to be identified by MS. Ulilysin (also called LysargiNase), a metalloprotease from Methanosarcina acetivorans, specifically cleaves at the N-terminal of arginine or lysine residues regardless of methylation status, − thus providing an alternative for proteomic profiling of cellular arginine methylation.…”

Tryp-N: A Thermostable Protease for the Production of N-terminal Argininyl and Lysinyl Peptides

“…12,13 A valuable addition to proteomic workflows, ulilysin produces peptides with the desired N-terminally localized basic centers. Moreover, this protein has been readily implemented in several proteomics workflows ranging from use as a simple substitute for trypsin in conventional workflows (reviewed by Giansanti et al 14 and Trevisiol et al 15 ) to improved characterization of protein termini, 16,17 enhanced protein identification, 18 and improved de novo peptide sequencing. 19,20 Given the utility of enzymes with this dual N-terminal specificity, we sought to find related enzymes, which could be similarly useful while presenting enhanced activity, specificity, and versatility.…”

“…Moreover, this protein has been readily implemented in several proteomics workflows ranging from use as a simple substitute for trypsin in conventional workflows (reviewed by Giansanti et al and Trevisiol et al , enhanced protein identification, and improved de novo peptide sequencing. , Given the utility of enzymes with this dual N-terminal specificity, we sought to find related enzymes, which could be similarly useful while presenting enhanced activity, specificity, and versatility. To that end, our approach was to informatically screen for putative proteases with favorable molecular features (described below), characterize their specificity, produce a top candidate via recombinant protein expression, and profile that candidateʼs activity under numerous biochemical conditions.…”