Christopher Hosfield (Madison, WI / US), Jessica Wohlfahrt (Tampa, FL / US), Jennifer Guergues (Tampa, FL / US), Marjeta Urh (Madison, WI / US), Michael Rosenblatt (Madison, WI / US), Stanley Stevens Jr. (Tampa, FL / US)
Introduction
In bottom-up proteomic studies, proteins are proteolytically digested into peptides that are subsequently identified using LC-MS/MS. Ideally, the protease used should be both highly efficient and specific, since poor digestion due to low specificity and/or efficiency complicates LC-MS/MS analysis by increasing sample complexity and reducing sensitivity. Here we describe the application of an ultra-efficient and ultra-specific protease that cleaves C-terminal to arginine residues and can produce near-zero missed-cleavage rates. This Arg-C Ultra protease can be used in combination with Lys-C to produce "tryptic" digests that are superior to those produce by trypsin or trypsin combined with Lys-C.
Methods
Human K562 extracts were digesed with combinations of Arg-C Ultra, Lys-C and Trypsin at a variety of digestion times and enzyme:substrate ratios to analyze digestion efficiency and specificity. Digested peptides were analyzed on an Orbitrap Exploris 240 (ThermoFisher) and data were searched with Byonic (Protein Metrics). Ubiquitination analysis utilized Protac-treated cells from which total protein was extracted, digested with Arg-C Ultra or Trypsin, followed by C18 cleanup and subsequent enrichment of peptides with the PTMScan HS Ubiquitin remnant motif (K-Ɛ-GG) magnetic beads (Cell Signaling). Histones were extracted from adult-derived immortalized mouse microglia (IMG cells) using the EpiQuik Total Histone Extraction Kit (EpiGentek). Samples were reduced before 2hr digestion with Arg-C Ultra followed by alkylation and desalting. Histone data were collected using a Bruker timsTOF Pro instrument and analyzed with FragPipe (using LFQ-MBR and open search workflows).
Results
Digestion of human K562 cell extract with Arg-C Ultra demonstrated superior cleavage efficiency and specificity over any protease tested to-date. The combination of Arg-C with Lys-C was shown to outperform Trypsin or Trypsin/Lys-C in terms of both digestion efficiency and specificity. Histone analysis was completed without prior protein and peptide derivatization (e.g., propionylation of lysine/N-termini). High sequence coverage of histones was obtained, which included the basic N-terminal tail regions of H2A, H2B, H3, and H4. Specifically, overall sequence coverage for the core histone variants was approximately 80% or greater (greatest at ~96% for H2B) with high digestion efficiency observed (<1% missed cleavages). Numerous PTMs were identified using either targeted or open search strategies including phosphorylation as well as lysine mono-, di-, tri-methylation, acetylation, formylation, ubiquitination (Gly-Gly tag), and butyrylation
Conclusions
Advanced proteomic sample preparation enabled by an ultra-efficient arginine-specific protease.
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