Proteomics

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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

ABSTRACT: 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.

OTHER RELATED OMICS DATASETS IN: MODEL1109130000MODEL1909260003MODEL1909260004MODEL1703310000MODEL1909260005MODEL1909260006MODEL1311110000MODEL1311110001

INSTRUMENT(S): Orbitrap Fusion, Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell, Cell Culture

SUBMITTER: Liana Tsiatsiani  

LAB HEAD: Albert JR Heck

PROVIDER: PXD004447 | Pride | 2016-12-02

REPOSITORIES: Pride

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Dataset's files

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Action DRS
C-ions_OT.pdStudy Other
C-ions_OT.pdStudy.bak Other
C-ions_OT.pdStudy.temp.bak Other
F1_Agilent5_20150701_LT_JurKR_ETciD30_2_1.msf Msf
F1_Agilent5_20150701_LT_JurKR_ETciD30_2_1.msfView Msf
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Publications

Opposite Electron-Transfer Dissociation and Higher-Energy Collisional Dissociation Fragmentation Characteristics of Proteolytic K/R(X)<sub>n</sub> and (X)<sub>n</sub>K/R Peptides Provide Benefits for Peptide Sequencing in Proteomics and Phosphoproteomics.

Tsiatsiani Liana L   Giansanti Piero P   Scheltema Richard A RA   van den Toorn Henk H   Overall Christopher M CM   Altelaar A F Maarten AF   Heck Albert J R AJ  

Journal of proteome research 20161202 2

A key step in shotgun proteomics is the digestion of proteins into peptides amenable for mass spectrometry. Tryptic peptides can be readily sequenced and identified by collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD) because the fragmentation rules are well-understood. Here, we investigate LysargiNase, a perfect trypsin mirror protease, because it cleaves equally specific at arginine and lysine residues, albeit at the N-terminal end. LysargiNase peptides are t  ...[more]

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