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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

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): Hermarchus Sp. Her2

SUBMITTER: Hao Yang

LAB HEAD: Ping Xu

PROVIDER: PXD008690 | Pride | 2019-01-11

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	A019_trypsin.raw	Raw
	AF019_lysarginase.raw	Raw
	AF024_LysargiNase_1.raw	Raw
	AF024_trypsin.raw	Raw
	pFind_AF019_Ac-LysargiNase.protein	Other

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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

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

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

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

Mirror proteases of Ac-Trypsin and Ac-LysargiNase precisely improve novel event identifications in Mycolicibacterium smegmatis MC2 155 by proteogenomic analysis.

Project description:Accurate identification of novel peptides remains challenging because of the lack of evaluation criteria in large-scale proteogenomic studies. Mirror proteases of trypsin and lysargiNase can generate complementary b/y ion series, providing the opportunity to efficiently assess authentic novel peptides in experiments other than filter potential targets by different false discovery rates (FDRs) ranking. In this study, a pair of in-house developed acetylated mirror proteases, Ac-Trypsin and Ac-LysargiNase, were used in Mycolicibacterium smegmatis MC2 155 for proteogenomic analysis. The mirror proteases accurately identified 368 novel peptides, exhibiting 75-80% b and y ion coverages against 65-68% y or b ion coverages of Ac-Trypsin (38.9% b and 68.3% y) or Ac-LysargiNase (65.5% b and 39.6% y) as annotated peptides from M. smegmatis MC2 155. The complementary b and y ion series largely increased the reliability of overlapped sequences derived from novel peptides. Among these novel peptides, 311 peptides were annotated in other public M. smegmatis strains, and 57 novel peptides with more continuous b and y pairs were obtained for further analysis after spectral quality assessment. This enabled mirror proteases to successfully correct six annotated proteins' N-termini and detect 17 new coding open reading frames (ORFs). We believe that mirror proteases will be an effective strategy for novel peptide detection in both prokaryotic and eukaryotic proteogenomics.

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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.

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