<b>Recombinant acetylated trypsin demonstrates superior stability and higher activity than commercial products in quantitative proteomics studies</b>

Wu, Feilin; Zhao, Mingzhi; Zhang, Yao; Su, Na; Zhi, Xiao‐Yang; Xu, Ping

doi:10.1002/rcm.7535

Cited by 21 publications

(20 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In addition to SDS, this procedure also removes non‐protein components, such as lipids and nucleic acids. Thus, a novel strategy combining FASP and high‐resolution hybrid orbitrap mass spectrometry has been widely used in biological studies in recent years …”

Section: Summary Of Psm Peptide Protein Groups and Average Coveragmentioning

confidence: 99%

Modified filter-aided sample preparation (FASP) method increases peptide and protein identifications for shotgun proteomics

Wang

Chen

et al. 2016

Rapid Commun. Mass Spectrom.

View full text Add to dashboard Cite

show abstract

Section: Summary Of Psm Peptide Protein Groups and Average Coveragmentioning

confidence: 99%

Modified filter-aided sample preparation (FASP) method increases peptide and protein identifications for shotgun proteomics

Wang

Chen

et al. 2016

Rapid Commun. Mass Spectrom.

View full text Add to dashboard Cite

show abstract

Section: Resultsmentioning

confidence: 92%

“…Modifications of the lysine residues of the native trypsin (methylated or acetylated) can highly increase its activity, stability and resistance to autolysis . For the LysargiNase, Huesgen et al have shown that LysargiNase can digest methylated lysine and arginine residues, but not acetylated ones; we assumed that acetyl modification could stabilize and improve the performance of our recombinant LysargiNase . To test this hypothesis, an acetylated procedure was performed on the purified recombinant LysargiNase according to the method described previously and the obtained rAc‐LysargiNase was further purified by gel filtration chromatography (Figures A and 5B).…”

Section: Resultsmentioning

confidence: 99%

“…The MS 1 precursors and MS 2 fractions were both detected in the Orbitrap with a resolution of 30,000 and 7500 at 400 m/z , respectively. The obtained raw files were searched against the complete proteome sequences of S. cerevisiae obtained from UniProt (version from February, 2015, modified by replacing its original ubiquitin protein by a His‐Myc tagged ubiquitin) and analyzed by MaxQuant as described previously with some modifications. Briefly, cleavage site rules for LysargiNase (N terminus of residue K/R) were set up for specific search and the maximum number of missed cleavages was fixed at 2.…”

Section: Methodsmentioning

confidence: 92%

See 1 more Smart Citation

Recombinant expression, purification and characterization of acetylated LysargiNase from Escherichia coli with high activity and stability

Zhang

Zhao

Xiao

et al. 2019

Rapid Comm Mass Spectrometry

Self Cite

View full text Add to dashboard Cite

Rationale LysargiNase is a novel characterized metalloprotease that can cleave the N‐terminii of lysine or arginine residues. The peptides generated by LysargiNase are just mirrors to those generated by trypsin. These characteristics of LysargiNase provide a powerful tool for mass spectrometry (MS)‐based proteomics research. A highly active and stable LysargiNase produced by an easy and inexpensive method could greatly benefit proteomics research. Here, we report the soluble recombinant expression, purification and acetyl modification of LysargiNase in Escherichia coli. Methods The coding sequence of LysargiNase with an enterokinase cleavage site at the N‐terminus was inserted into plasmid pGEX‐4 T‐2 and transformed into E. coli BL21 (DE3). The strain was cultured in a 14‐L fermenter with a working volume of 5 L. The protein expression was induced by adding isopropyl‐β‐D‐thiogalactoside (IPTG) to a final concentration of 1 mM. The recombinant LysargiNase was loaded onto a GSTrap and an on‐column digestion was performed to remove the GST tag and was subsequently purified by chromatographic purification. In vitro acetylation of LysargiNase was performed by using acetic anhydride. The digestion efficiency and specificity of recombinant LysargiNase and acetylated LysargiNase were compared with simple protein substrate, human serum albumin (HSA), and a complex proteomic sample, yeast lysate, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). Results Highly soluble expression of recombinant LysargiNase was achieved by plasmid pGEX‐4 T‐2 in E. coli BL21 (DE3). In addition, acetylation of purified LysargiNase significantly increased its resistance to autolysis, which resulted in a more complete digestion of proteomics samples and more identified peptides and proteins by LC/MS/MS. Conclusions In this study, we constructed a highly soluble expression system for producing recombinant LysargiNase in E. coli, which gave tremendous advantages in the downstream purification process. We also confirmed that acetyl modification can increase the stability and activity of recombinant LysargiNase. The study provided a superior way to produce this powerful tool for proteomics research.

show abstract

“…In‐solution digestion was performed as described previously . SDS was removed by a 10 kDa filtration unit .…”

Section: Methodsmentioning

confidence: 99%

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

Liu

Yin

et al. 2019

Rapid Comm Mass Spectrometry

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Recombinant acetylated trypsin demonstrates superior stability and higher activity than commercial products in quantitative proteomics studies

Abstract: The r-Ac-trypsin described here is a recombinant product. In addition it showed similar or superior properties such as stability activity and specificity to commercial products. It can be used in peptide sample preparation in proteomics studies.

Cited by 21 publications

References 27 publications

Modified filter-aided sample preparation (FASP) method increases peptide and protein identifications for shotgun proteomics

Modified filter-aided sample preparation (FASP) method increases peptide and protein identifications for shotgun proteomics

Recombinant expression, purification and characterization of acetylated LysargiNase from Escherichia coli with high activity and stability

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

Contact Info

Product

Resources

About