Unambiguous Phosphosite Localization through the Combination of Trypsin and LysargiNase Mirror Spectra in a Large-Scale Phosphoproteome Study

Abstract

Understanding of the kinase-guided signaling pathways requires the identification and analysis of phosphosites. Mass spectrometry (MS)-based phosphoproteomics is a rapid and highly sensitive approach for high-throughput identification of phosphosites. However, phosphosite determination from MS data with a single protease is more likely to be ambiguous, regardless of the strategy used for phosphopeptide detection. Here, we 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. We found that the combination of trypsin and LysargiNase mirror spectra resulted in higher ion coverage in MS2 spectra. The median ion coverage values of b ions in tryptic spectra, LysargiNase spectra, and combined spectra are 8.3, 20.5, and 25.0%, respectively. As for the median ion coverage of y ions, these values are 27.8, 10.0, and 32.3%. Higher ion coverage was helpful to pinpoint the precise phosphosites. Compared to trypsin alone, the combined use of trypsin and LysargiNase mirror spectra enabled 67.1% of mirror spectra with unreliable scores (confidence score <0.75) to become reliable (confidence score ≥ 0.75). Meanwhile, all of the mirror peptide-spectrum matches (PSMs) with multiple potential phosphosites from trypsin and LysargiNase digests could be assigned one precise phosphosite after applying the combination strategy. Besides, the combination strategy could identify more novel phosphosites than the union strategy did. We synthesized three phosphopeptides corresponding to the three novel phosphosites and validated the reliability of the identification. Taken together, our data demonstrated the distinctive potential of the combination strategy presented here for unambiguous phosphosite localization (Project accession PXD011178).

ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates

“…Ulilysin has been compared with trypsin from various perspectives in previously reported proteomic studies. ,, First of all, proteolysis by ulilysin generates peptides with N-terminal lysine or arginine residues, which result in strong b-ion series during CID and c-ion series during ETD fragmentation, improving peptide sequencing . In addition, unlike trypsin, cleavage by ulilysin occurs at monomethylated and dimethylated lysine and arginine, facilitating the detection of these important PTMs .…”

The phosphoproteome is a first responder in tiered cellular adaptation to chemical stress followed by proteomics and transcriptomics alteration

“…Tryptic peptides were desalted by reverse-phase C18 Sep-Pak extraction cartridges (Waters Corporation, Milford, MA, USA). Cleaned peptides were resuspended in buffer A (1% ACN, 0.1% trifluoroacetic acid) and separated into 12 fractions through a Bonna-Agela C18 column (5 µm reverse-phase fused-silica, 4.6 × 250 mm column) on a RIGOL-L3000 HPLC (RIGOL, Beijing, China).The phosphor-peptides in each fraction were enriched with a Ti 4+ -immobilized metal ion affinity chromatography (IMAC) method as previously described with slight modification 44, 45 . Briefly, dried peptides were dissolved in 500 μL binding buffer (80% ACN, 6% trifluoroacetic acid (TFA) in ddH 2 O) and the same volume of loading buffer (10% 500 mM NH 4 HCO 3 , 5% ACN in ddH 2 O) followed by addition of 25 mg Ti 4+ -IMAC beads.…”

Using NAMs to characterize chemical bioactivity at the transcriptomic, proteomic and phosphoproteomic levels

“…For proteomics and phosphoproteomics, the same HepG2 cells were recovered immediately after 10 min and 24 h of caffeine treatment. Proteins were then digested in-gel with acetylated trypsin 31, 32 and LysC at 37 °C for 14 h. Subsequently, peptides were enriched with the Ti 4+ -immobilized metal ion affinity chromatography (IMAC) method, as previously described 33, 34 . The peptides of total cell lysate and enriched phoshopeptides were separate into 3 fractions and then detected by Q-Exactive HF mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) and LUMOS mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) with a data dependent acquisition (DDA) mode.…”