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Characterization of LysargiNase for use in phosphoproteomics experiments, partII

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

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Pitter Huesgen

LAB HEAD: Christopher M. Overall

PROVIDER: PXD001114 | Pride | 2014-12-16

REPOSITORIES: Pride

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LysargiNase mirrors trypsin for protein C-terminal and methylation-site identification.

Huesgen Pitter F PF Lange Philipp F PF Rogers Lindsay D LD Solis Nestor N Eckhard Ulrich U Kleifeld Oded O Goulas Theodoros T Gomis-Rüth F Xavier FX Overall Christopher M CM

Nature methods 20141124 1

To improve proteome coverage and protein C-terminal identification, we characterized the Methanosarcina acetivorans thermophilic proteinase LysargiNase, which cleaves before lysine and arginine up to 55 °C. Unlike trypsin, LysargiNase-generated peptides had N-terminal lysine or arginine residues and fragmented with b ion-dominated spectra. This improved protein C terminal-peptide identification and several arginine-rich phosphosite assignments. Notably, cleavage also occurred at methylated or di ...[more]

PMID: 25419962

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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

LysargiNase and tryptic digest of MDA-MB 231 cell lysates

Project description:Comparison of peptide properties in proteomes digested with LysargiNase in comparison to trypsin

2014-12-16 | PXD001113 | Pride

LysargiNase cleavage specificity characterization

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

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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.

2016-12-02 | PXD004447 | Pride

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

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

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

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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

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

2019-01-11 | PXD008690 | Pride

Human Testis LC-MS/MS - Multi-Protease Strategy Identifies Three PE2 Missing Proteins in Human Testis Tissue

Project description:Missing proteins(MPs) investigation is one of the critical missions of Chromosome-based Human Proteome Project(C-HPP). Through analyzing sequence of MPs from neXtProt database, we found that there are no detectable tryptic peptides for some MPs. Here, we attmept a multi-enzymes strategy to cover this problem. Lys-C, Glu-C and LysargiNase are used to produce more unique and detectable peptides of MPs.

2017-10-09 | PXD006465 | 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

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