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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

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

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Saccharomyces Cerevisiae (strain Atcc 204508 / S288c) (baker's Yeast)

SUBMITTER: Xi Wang

LAB HEAD: Ping Xu

PROVIDER: PXD011562 | Pride | 2019-01-11

REPOSITORIES: Pride

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			Action	DRS
	R29_LC_1.raw	Raw
	R29_LC_10.raw	Raw
	R29_LC_11.raw	Raw
	R29_LC_12.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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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

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

LysargiNase cleavage specificity characterization

Project description:Determination of LysargiNase and tryptic ckeavage efficiency at dimethylated Lys residues

2014-12-16 | PXD001379 | Pride

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

Project description: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 algorithm, pNovoM, for de novo sequencing. Using pNovoM to sequence peptides of purified proteins, the accuracy of the sequence was close to 100%. More importantly, from a large-scale yeast proteome sample digested with trypsin and Ac-LysargiNase individually, 48% of all tandem mass spectra formed mirror spectrum pairs, 97% of which contained full coverage of ion series, resulting in precision de novo sequencing of full-length peptides by pNovoM. This enabled pNovoM to successfully sequence 21,249 peptides from 3,753 proteins and interpreted 44-152% more spectra than pNovo+ and PEAKS at a 5% FDR at the spectrum level. Moreover, the mirror protease strategy had an obvious advantage in sequencing long peptides. We believe that the combination of mirror protease strategy and pNovoM will be an effective approach for precision de novo sequencing on both single proteins and proteome samples.

| S-EPMC6442358 | biostudies-literature

Specificity profiling of LysargiNase in comparison to trypsin

Project description:Protease cleavage site preferences of LysargiNase (former name: ulilysin) and trypsin were tested using proteome-derived peptide libraries (Schilling et al Nature Protocols 2011)

2014-12-16 | PXD001122 | 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

Characterization of LysargiNase for use in phosphoproteomics experiments, part 1

Project description:Use of parallel digest with LysargiNase (former name: ulilysin) and trypsin to cover complementary phosphosites / characterization of phospho-motifs preferentially identified by each protease

2014-12-16 | PXD001121 | Pride

Characterization of LysargiNase for use in phosphoproteomics experiments, partII

Project description:Use of parallel digest with LysargiNase and trypsin to cover complementary phosphosites / characterization of phospho-motifs preferentially identified by each protease

2014-12-16 | PXD001114 | Pride

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.

2020-05-22 | PXD011178 | Pride