Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Mirror-image trypsin digestion and sequencing of D-proteins

ABSTRACT: The development of mirror-image biology systems and related applications is hindered by the lack of effective methods to sequence mirror-image (D-) proteins. Although natural-chirality (L-) proteins can be sequenced by bottom–up liquid chromatography–tandem mass spectrometry (LC–MS/MS), the sequencing of long D-peptides and D-proteins with the same strategy requires digestion by a site-specific D-protease before mass analysis. Here we apply solid-phase peptide synthesis and native chemical ligation to chemically synthesize a mirror-image version of trypsin, a widely used protease for site-specific protein digestion. Using mirror-image trypsin digestion and LC–MS/MS, we sequence a mirror-image large subunit ribosomal protein (L25) and a mirror-image Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), and distinguish between different mutants of D-Dpo4. We also perform writing and reading of digital information in a long D-peptide of 50 amino acids. Thus, mirror-image trypsin digestion in conjunction with LC–MS/MS may facilitate practical applications of D-peptides and D-proteins as potential therapeutic and informational tools.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Escherichia Coli

SUBMITTER: Guanwei Zhang

LAB HEAD: Ting Zhu

PROVIDER: PXD046228 | Pride | 2024-01-19

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

Expression data from mammary epithelial cells harvested from 8-9 week old C3H female mice - various isolation conditions

2014-09-05 | GSE61167 | GEO

E. coli mirror LC-MS/MS - Precision de novo peptide sequencing using mirror proteases of Ac-LysargiNase and trypsin for large-scale proteomics

Project description:Precision de novo peptide sequencing using mirror proteases of Ac-LysargiNase and trypsin for large-scale proteomicsPrecision de novo peptide sequencing using mirror proteases of Ac-LysargiNase and trypsin for large-scale proteomics

2019-01-11 | PXD008688 | Pride

Monoclonal antibody LC-MS/MS - Precision de novo peptide sequencing using mirror proteases of Ac-LysargiNase and trypsin for large-scale proteomics

Project description:Precision de novo peptide sequencing using mirror proteases of Ac-LysargiNase and trypsin for large-scale proteomics

2019-01-11 | PXD008690 | Pride

Yeast mirror LC-MS/MS - Precision de novo peptide sequencing using mirror proteases of Ac-LysargiNase and trypsin for large-scale proteomics

Project description:Precision de novo peptide sequencing using mirror proteases of Ac-LysargiNase and trypsin for large-scale proteomics

2019-01-11 | PXD011562 | Pride

Expression data from mammary epithelial cells harvested from 8-9 week old C3H female mice - various isolation conditions

Project description:Numerous lineage analysis studies have used FACS sorting as a separation technique prior to measuring mRNA expression patterns. To assess if FACS sorting causes non-specific changes in gene expression, we collected a heterogeneous population of mammary epithelial cells from three biological replicates. We then performed gene expression analysis on each replicate either prior to trypsin digestion, immediately following trypsin digestion, or following both the trypsin digestion and a mock FACS sort. In this dataset, we include gene expression data obtained from isolated murine mammary epithelial cells either prior to trypsin digestion, immediately following trypsin digestion, or following the trypsin digestion and a mock FACS sort. Nine total samples were analyzed (3 biological replicates of three experimental conditions). Using LIMMA packages gene-wise comparisons were made between untrypsinized and trypsinized replicates as well as between trypsinized and mock FACS sorted replicates using and Pâ¤0.05 and a â¥1.5-fold difference between conditions.

2014-09-05 | E-GEOD-61167 | biostudies-arrayexpress

Mirroring the charged termini; ETD/HCD fragmentation characteristics of LysargiNase and tryptic peptides and their benefits for peptide sequencing in proteomics

Project description:A key step in proteomics is the digestion of proteins into peptides, so far largely done by using trypsin. Tryptic digestion leads to peptides that in ESI-MS attain predominantly two charges, via protonation at the free N-terminus and at the C-terminal basic residue Arginine or Lysine. These peptides can be readily sequenced and identified by collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD), as the fragmentation rules are well understood. Here we explore the trypsin mirror protease, LysargiNase, which cleaves equally specifically at Arg and Lys, albeit at the N-terminal end. The resulting peptides are therefore practically tryptic-alike in length and sequence, except that the two charges are now both positioned at the N-terminus. We compare the chromatographic separation properties, gas phase fragmentation behavior and (phospho)proteome sequence coverage of tryptic and LysargiNase peptides using electron-transfer dissociation (ETD), and for comparison HCD. We find that tryptic and LysargiNase peptides fragment nearly as mirror images. For LysargiNase predominantly N-terminal peptide ions (c-ions/ETD, b-ions/HCD) are formed, whereas for trypsin C-terminal fragment ions dominate (z-ions/ETD, y-ions/HCD). Especially during ETD LysargiNase peptides fragment into low-complexity, but information rich sequence ladders. We observe that trypsin and LysargiNase chart distinct parts of the (phospho)proteome. Therefore, we conclude that the collective use LysargiNase and Trypsin will benefit a more in-depth and reliable analysis of (phospho)proteomes.

2016-12-02 | PXD004447 | Pride

2018CHPP - Digging for Missing Proteins Using Low-Molecular-Weight Protein Enrichment and a "Mirror Protease" Strategy

Project description:ABSTRACT Since 2012, Chromosome-Centric Human Proteome Project (C-HPP) launched the journey of missing proteins (MPs) investigation for completing human proteome project (HPP). We found the majority of MPs were distributed in low molecular weight (LMW) ranges, especially in 0-40 kDa. The identification of LMW proteins is challenging owing to their low length, low abundancy, and hydrophobicity. Even worse, many sequences from Trypsin digestion are unlikely to yield detectable peptides or composed less number of observable peptides in MS detection. Therefore, we focused on small MPs by combining LMW protein enrichment and mirror-protease strategy on human testis samples. The in-depth testis LMW protein profiling documented 4,063 proteins from which 2,565 proteins were belong to LMW proteins and 1,130 proteins had mirror peptides. This provided mass spectral evidence for small MPs further verification, and two MPs were verified in seven identified MPs. For Q8N688, confident peptides were verified from Trypsin and LysargiNase digestions, and continuous b/y-ion peptide was obtained by mirror-spectrum matching. Two verified peptides of Q86WR6 were from gel-separation and gel-elution dataset, respectively. And the other 5 MPs cannot be distinguished sufficiently as PE1, but need more verification information.

2018-10-04 | PXD010093 | Pride

Proteomics Using Proteases Alternative to Trypsin Benefits from Sequential Digestion with Trypsin

Project description:High specificity and ease of use make trypsin the most used enzyme in proteomics. Proteases with complementary cleavage specificity to trypsin have been applied to obtain additional data. However, use of proteases with broad specificity proved especially challenging. In this work, we analyzed the characteristics of five protease alternatives to trypsin for protein identification and sequence coverage when applied to S. pombe whole cell lysates. The specificity of the protease heavily impacted on the number of proteins identified. Proteases with higher specificity let to the identification of more proteins than proteases with lower specificity. However, AspN, GluC, chymotrypsin and proteinase K largely benefited from being paired with trypsin in sequential digestion, as had been shown by us for elastase before. In the most extreme case, the addition of trypsin to a proteinase K digest increased the number of identified proteins by 524 %. Also, AspN (82 %) and GluC (74 %) protein identifications largely improved following the additional digestion with trypsin. In general, protein identifications improved most over the use of the single protease when the enzymes followed on an initial digestion with trypsin. In the most extreme case, the sequential digest with trypsin and AspN yielded even higher number of protein identifications than digesting with trypsin alone.

2021-06-07 | PXD017321 | Pride

Perception of fight outcome is needed to activate socially driven changes in brain transcriptome

Project description:Group living animals must be able to express different behavior profiles depending on their social status. This implies that the same genotype may translate into different behavioral phenotypes through socially driven differential gene expression. Here we show for the first time that what triggers the switch between status-specific neurogenomic states is not the objective structure of the social interaction but rather the subjectsM-bM-^@M-^Y perception of its outcome. For this purpose we had male zebrafish fight either a real opponent or their own image on a mirror. Massive changes in the brain transcriptome were observed in real opponent fighters, which experience either a victory or a defeat. In contrast, mirror fighters, which had no information on fight outcome despite expressing aggressive behavior, failed to activate a neurogenomic response. These results indicate that, even in cognitively simple organisms such as zebrafish, neurogenomic responses underlying changes in social status rely on cognitive appraisal. Brain samples from zebrafish, Danio rerio were collected after experimental manipulations generated 4 phenotypes regarding social experience: mirror fighters (M), winners of a real opponent fight (W), losers of a real opponent fight (L); and socially isolated fish (I). After 30 minutes of experimental manipulation animals were anesthetized with a lethal dose of MS-222 (1000-1500 mg/l) and decapitated. Brains were rapidly collected, homogenized and RNA extracted following standard methods of RNeasy Lipid Tissue Mini Kit, Qiagen. RNA was then treated with DNase (RNase-free DNase set, Qiagen) to remove possible contaminations with genomic DNA and concentration and purity was estimated by spectrophotometric absorbance in a NanoDrop ND-1000 UV-Vis Spectrophotometer (Nano-Drop Technologies).

2014-04-10 | E-GEOD-56549 | biostudies-arrayexpress

Small non-coding RNA expression in murine germ cells

Project description:In order to monitor the changes in small RNAs expression during mouse spermatogenesis. Type A spermatogonia, pachytene spermatocytes and round spermatids were isolated following collagenase treatment of testes and trypsin digestion of isolated seminiferous tubules using unit gravity sedimentation in a bovine serum albumin gradient. The small RNA fraction (18-36nt) was cloned and sequenced from total RNA of each cell type.

2011-06-10 | GSE24822 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data