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Tryp-N: A thermostable protease for the production of N-terminal argininyl and lysinyl peptides

ABSTRACT: Identification and characterization of Tryp-N, A thermostable protease for the production of N-terminal argininyl and lysinyl peptides

INSTRUMENT(S): LTQ Orbitrap XL

ORGANISM(S): Escherichia Coli

TISSUE(S): Cell Culture

SUBMITTER: Jonathan Ipsaro

LAB HEAD: Darryl J. Pappin

PROVIDER: PXD017021 | Pride | 2020-04-22

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	TrypN_pH10.RAW	Raw
	TrypN_pH10.dat	Other
	TrypN_pH11.RAW	Raw
	TrypN_pH11.dat	Other
	TrypN_pH12.RAW	Raw

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1 - 5 of 26

Publications

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

Wilson John P JP Ipsaro Jonathan J JJ Del Giudice Samantha N SN Turna Nikita Saha NS Gauss Carla M CM Dusenbury Katharine H KH Marquart Krisann K Rivera Keith D KD Pappin Darryl J DJ

Journal of proteome research 20200320 4

Bottom-up proteomics is a mainstay in protein identification and analysis. These studies typically employ proteolytic treatment of biological samples to generate suitably sized peptides for tandem mass spectrometric (MS) analysis. In MS, fragmentation of peptides is largely driven by charge localization. Consequently, peptides with basic centers exclusively on their N-termini produce mainly b-ions. Thus, it was long ago realized that proteases that yield such peptides would be valuable proteomic ...[more]

PMID: 32141294

Similar Datasets

Tryp-N: A thermostable protease for the production of N-terminal argininyl and lysinyl peptides

Project description:Identification and characterization of Tryp-N, A thermostable protease for the production of N-terminal argininyl and lysinyl peptides

2020-04-22 | PXD017030 | Pride

Tryp-N: A thermostable protease for the production of N-terminal argininyl and lysinyl peptides

Project description:Identification and characterization of Tryp-N, A thermostable protease for the production of N-terminal argininyl and lysinyl peptides

2020-04-22 | PXD017023 | Pride

Tryp-N: A thermostable protease for the production of N-terminal argininyl and lysinyl peptides

Project description:Identification and characterization of Tryp-N, A thermostable protease for the production of N-terminal argininyl and lysinyl peptides

2020-04-22 | PXD017024 | Pride

Tryp-N: A thermostable protease for the production of N-terminal argininyl and lysinyl peptides

Project description:Identification and characterization of Tryp-N, A thermostable protease for the production of N-terminal argininyl and lysinyl peptides

2020-04-22 | PXD017048 | Pride

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

Project description:Bottom-up proteomics is a mainstay in protein identification and analysis. These studies typically employ proteolytic treatment of biological samples to generate suitably sized peptides for tandem mass spectrometric (MS) analysis. In MS, fragmentation of peptides is largely driven by charge localization. Consequently, peptides with basic centers exclusively on their N-termini produce mainly b-ions. Thus, it was long ago realized that proteases that yield such peptides would be valuable proteomic tools for achieving simplified peptide fragmentation patterns and peptide assignment. Work by several groups has identified such proteases, however, structural analysis of these suggested that enzymatic optimization was possible. We therefore endeavored to find enzymes that could provide enhanced activity and versatility while maintaining specificity. Using these previously described proteases as informatic search templates, we discovered and then characterized a thermophilic metalloprotease with N-terminal specificity for arginine and lysine. This enzyme, dubbed Tryp-N, affords many advantages including improved thermostability, solvent and detergent tolerance, and rapid digestion time.

| S-EPMC7842235 | biostudies-literature

Introducing synthetic thermostable RNase inhibitors in single-cell RNA-seq

Project description:Single-cell RNA-sequencing (scRNAseq) is revolutionizing biomedicine, propelled by advances in methodology, ease of use, and cost reduction of library preparation. Over the past decade, there have been remarkable technical improvements in most aspects of single-cell transcriptomics. Yet, there has been little to no progress in advancing RNase inhibition despite that maintained RNA integrity is critical during cell collection, storage, and cDNA library generation. Here, we demonstrate that a synthetic thermostable RNase inhibitor yield single-cell libraries of equal or superior quality compared to ubiquitously used protein-based recombinant RNase inhibitors (RRIs). Importantly, the synthetic RNase inhibitor provide additional unique improvements in reproducibility and throughput, enable new experimental workflows including heat cycles, and can reduce the need for dry-ice transports. In summary, replacing RRIs represents a substantial advancement in the field of single-cell transcriptomics.

2024-07-29 | E-MTAB-13873 | biostudies-arrayexpress

Introducing synthetic thermostable RNase inhibitors in single-cell RNA-seq (Bulk RNA-seq libraries)

2024-07-26 | E-MTAB-13899 | biostudies-arrayexpress

Production of thermostable phycocyanin in a mesophilic cyanobacterium.

Project description:Phycocyanin (PC) is a soluble phycobiliprotein found within the light-harvesting phycobilisome complex of cyanobacteria and red algae, and is considered a high-value product due to its brilliant blue colour and fluorescent properties. However, commercially available PC has a relatively low temperature stability. Thermophilic species produce more thermostable variants of PC, but are challenging and energetically expensive to cultivate. Here, we show that the PC operon from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (cpcBACD) is functional in the mesophile Synechocystis sp. PCC 6803. Expression of cpcBACD in an 'Olive' mutant strain of Synechocystis lacking endogenous PC resulted in high yields of thermostable PC (112 ± 1 mg g-1 DW) comparable to that of endogenous PC in wild-type cells. Heterologous PC also improved the growth of the Olive mutant, which was further supported by evidence of a functional interaction with the endogenous allophycocyanin core of the phycobilisome complex. The thermostability properties of the heterologous PC were comparable to those of PC from T. elongatus, and could be purified from the Olive mutant using a low-cost heat treatment method. Finally, we developed a scalable model to calculate the energetic benefits of producing PC from T. elongatus in Synechocystis cultures. Our model showed that the higher yields and lower cultivation temperatures of Synechocystis resulted in a 3.5-fold increase in energy efficiency compared to T. elongatus, indicating that producing thermostable PC in non-native hosts is a cost-effective strategy for scaling to commercial production.

| S-EPMC8209669 | biostudies-literature

Production, Purification, and Characterization of Thermostable Alkaline Xylanase From Anoxybacillus kamchatkensis NASTPD13.

Project description:Anoxybacillus kamchatkensis NASTPD13 used herein as a source for thermostable alkaline xylanase were isolated from Paudwar Hot Springs, Nepal. NASTPD13 cultured at 60°C, pH 7 and in presence of inorganic (ammonium sulfate) or organic (yeast extract) nitrogen sources, produced maximum xylanase enzyme. Xylanase production in the cultures was monitored by following the ability of culture media to hydrolyze beech wood xylan producing xylooligosaccharide and xylose by thin layer chromatography (TLC). The extracellular xylanase was isolated from optimized A. kamchatkensis NASTPD13 cultures by ammonium sulfate (80%) precipitation; the enriched xylanase preparation was dialyzed and purified using Sephadex G100 column chromatography. The purified xylanaseshowed 11-fold enrichment with a specific activity of 33 U/mg and molecular weight were37 kDa based on SDS-PAGE and PAGE-Zymography. The optimum pH and temperature of purified xylanase was 9.0 and 65°C respectively retainingmore than 50% of its maximal activity over a broad range of pH (6-9) and temperature (30-65°C). With beech wood xylan, the enzyme showed Km 0.7 mg/ml and Vmax 66.64 ?M/min/mg The xylanase described herein is a secretory enzyme produced in large quantities by NASTPD13 and is a novel thermostable, alkaline xylanase with potential biotechnological applications.

| S-EPMC5962792 | biostudies-literature

Crosslinking experiment of the RAGE-S100B complex

Project description:XL-MS of RAGE-100B complex. The Crosslinking agent is BS3. Samples were subjected to sequential digestion. 91212_AM_2_2_1_HCDFT, 91212_AM_2_2_2_HCDFT, 91212_AM_5_2_2_HCDFT, 91212_AM_5_2_2_HCDFT - Tryp-Chym; 91212_AM_2_3_1_HCDFT, 91212_AM_2_3_2_HCDFT, 91212_AM_5_3_2_HCDFT, 91212_AM_5_3_2_HCDFT - Tryp-Gluc; 91212_AM_2_1_1_HCDFT, 91212_AM_2_1_2_HCDFT, 91212_AM_5_1_2_HCDFT, 91212_AM_5_1_2_HCDFT - Tryp-Tryp.

2020-11-11 | MSV000086437 | MassIVE

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