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

ABSTRACT: 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

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Escherichia Coli

SUBMITTER: Hao Yang

LAB HEAD: Ping Xu

PROVIDER: PXD008688 | Pride | 2019-01-11

REPOSITORIES: Pride

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			Action	DRS
	UPLC_gel_lysargiNase_01.raw	Raw
	UPLC_gel_lysargiNase_02.raw	Raw
	UPLC_gel_lysargiNase_03.raw	Raw
	UPLC_gel_lysargiNase_04.raw	Raw
	UPLC_gel_lysargiNase_05.raw	Raw

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Publications

Precision De Novo Peptide Sequencing Using Mirror Proteases of Ac-LysargiNase and Trypsin for Large-scale Proteomics.

Yang Hao H Li Yan-Chang YC Zhao Ming-Zhi MZ Wu Fei-Lin FL Wang Xi X Xiao Wei-Di WD Wang Yi-Hao YH Zhang Jun-Ling JL Wang Fu-Qiang FQ Xu Feng F Zeng Wen-Feng WF Overall Christopher M CM He Si-Min SM Chi Hao H Xu Ping P

Molecular & cellular proteomics : MCP 20190108 4

De novo peptide sequencing for large-scale proteomics remains challenging because of the lack of full coverage of ion series in tandem mass spectra. We developed a mirror protease of trypsin, acetylated LysargiNase (Ac-LysargiNase), with superior activity and stability. The mirror spectrum pairs derived from the Ac-LysargiNase and trypsin treated samples can generate full b and y ion series, which provide mutual complementarity of each other, and allow us to develop a novel ...[more]

PMID: 30622160

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

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

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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.

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Mirror-image trypsin digestion and sequencing of D-proteins

Project description: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.

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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.

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Multiple de novo copy number variant (MdnCNV) driven mirror traits and blended phenotype

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Multiple de novo copy number variant (MdnCNV) driven mirror traits and blended phenotype

Project description:Purpose: To determine the genetic variation underlying phenotypic trait manifestation in a Multiple de novo copy number variant (MdnCNV) case. Method: Clinical microarray identified a previously unreported MdnCNV proband. Short and long-read genomic sequencing, SV analyses, and visualization tools for merged datasets were implemented to characterize MdnCNV mutagenesis. Human phenotype ontology based quantitative analyses identified gene(s) driving proband phenotype.Results: Eight dnCNVs of average length ~1 Mb were mapped in the MdnCNV proband. Sequence microhomology/microhomeology was present at 6/8 breakpoint junctions. dnSNVs (6/79) and de novo indels (1/12) are enriched within 4 Mb of the dnCNV regions. Of the duplication encompassed genes, NSD1 and SMARCC2 had the highest phenotypic match to the proband. Conclusion: Biparental origin of constitutive dnCNVs (4:4) supports an early perizygotic mutagenesis event as the cause of genome-wide hypermutation. Microhomology/microhomeology and regional hypermutation supports microhomology-mediated break-induced replication as the process underlying MdnCNV formation. The family reported here, together with published NSD1 variation associated cases further define NSD1 as a triplosensitivity locus that exhibits mirror quantitative traits from those associated with its haploinsufficiency. Quantitative phenotype analysis identified NSD1 and SMARCC2 as contributors to a blended phenotype.

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The use of LysargiNase complementary to trypsin in large scale phosphoproteome study for unambiguous phosphosite localization

Project description:Understanding of kinase-guided signaling pathways requires identification and analysis of phosphorylation sites. Mass spectrometry (MS)-based phosphoproteomics is a rapid and highly sensitive approach for high-throughput identification of phosphorylation sites. However, the exact localization of phosphorylation sites which depends on trypsin-centric shotgun phosphoproteomics is often arbitrary and unreliable because of the limited product ion coverage in the MS2 spectra. Therefore, phosphorylation site determination from MS data with a single protease cannot be expected to be comprehensive, regardless of the strategy used for phosphopeptide detection. Here, we have explored the application of LysargiNase which was recently reported to mirror trypsin in specificity to cleave arginine and lysine residues exclusively at the N-terminal side. Our data demonstrates that the combined application of LysargiNase and trypsin results in higher sequence coverage with more complete ladder sequences which is helpful to pinpoint the precise phosphorylation sites. Therefore, the combined use of these two enzymes will partially overcome the limitations of trypsin-centric phosphoproteomics to achieve a more reliable phosphoproteome and to increase the identification of novel phosphorylation sites.

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