Temporal regulation of Lsp1 O-GlcNAcylation and phosphorylation duringactivated Bcellsapoptosis
Here, we decipher the molecular mechanisms bridging B cell activation and apoptosis mediated by post-translational modification(PTM). We found that O-GlcNAcase inhibition enhances B cell activation and apoptosisinduced by B cell receptor (BCR)cross-linking. This proteome-scale analysis of the functional interplay between protein O-GlcNAcylation and phosphorylation in stimulated mouse primary B cells identified313O-GlcNAcylation-dependent phosphositeson 224 phosphoproteins.Among these phosphoproteins, temporal regulation ofthe O-GlcNAcylation and phosphorylation of lymphocyte-specific protein-1 (Lsp1) is a key switch that triggersapoptosis in activated B cells. O-GlcNAcylation at S209 of Lsp1, identified as the major O-GlcNAc site on Lsp1 by both electron-transfer dissociationand collision-induced dissociationtandem mass spectrometry,was a prerequisite for the recruitment of its kinase, PKC-β1, to induce S243 phosphorylation, leading to ERK activation and down-regulation of BCL-2 and BCL-xL. Thus, we demonstrate the critical PTM interplay of Lsp1 that transmits signals for initiating apoptosis after BCR ligation.
Sample Processing Protocol
For packing IMAC column, a 10 cm microcolumn (500 μm id PEEK column, Upchurch Scientific/Rheodyne) that has been packed with Ni-NTA resin (Qiagen)was enclosed in a stainless steel column-end fitted with a 0.5μm frit disk at one end.Phosphopeptide purification was performed using an autosampler and an HP1100 solvent delivery system (Hewlett-Packard). The flow rate was set at 13μlmin-1and the loading/condition buffer was 6% (v/v) AA with pH adjusted to 3.0. Ni2+ions on the resin were removed by an amount of 100 μlof 50 mM EDTA in 1 M NaCl followed by equilibrationwith loading buffer for 15 min. To equip the IMAC column with Fe3+, 100 μl of 0.2 M FeCl3was loaded into the column for another 15 min before sample loading. Peptide samples were resolved in loading buffer and loaded into theFe3+-equipped IMAC column for 20 min. A total of 100 μlof 25% (v/v) ACN was used to wash the unbound peptides away for 15 min. The bound peptides were eluted with 100 μl of 200 mM NH4H2PO4 and dried by vacuum centrifugation for further use. LC-MS/MS analysis was performed on an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) for phosphoproteome which equipped with a nanospray interface (Proxeon, Odense, Denmark). Peptides were separated on a nanoAcquity system (Waters, Milford, MA) which was connected to mass spectrometry. Peptide mixtures were loaded onto a 75 μm ID, 25 cm length C18 BEH column (Waters, Milford, MA) packed with 1.7 μm particles with a pore of 130 Å. A segmented gradient in 90 min from 1% to 35% solvent B (acetonitrile with 0.1% formic acid) at a flow rate of 300 nl min-1 and a column temperature of 35°C were used. Solvent A was 0.1% formic acid in water. Survey full scan MS spectra were acquired in the orbitrap (m/z 350–1600) with the resolution set to 60,000 at m/z 400 and automatic gain control (AGC) target at 106. The mass spectrometer was operated in the data-dependent mode.The 10 most intense ions were sequentially isolated for collision activated dissociation (CID) MS/MS fragmentation and detected in the linear ion trap (AGC target at 7000) with previously selected ions dynamically excluded for 90 s. Ions with singly and unrecognized charge state were excluded. To improve the fragmentation spectra of the phosphopeptides, “multistage activation” at 97.97, 48.99, and 32.66 Thomson (Th) relative to the precursor ion was enabled in all MS/MS events. All the measurements in the Orbitrap were performed with the lock mass option for internal calibration.
Data Processing Protocol
Raw MS/MS data were converted into peak lists (MGF file) using Raw2MSM with default parameters. The resulting peak lists were searched against theIPI_MOUSE_3.87 database (version 3.87, 68161 entries) via an in-house Mascot search engine (Matrix Science Ltd., UK; version 2.2.1). The search parameters were set as follows: peptide mass tolerance, 10 ppm Da; MS/MS ion mass tolerance, 0.6 Da; enzyme set as trypsin and allowance of up to two missed cleavages; variable modifications included oxidation on methionine and phosphorylation on serine, threonine, and tyrosine residues; peptide charge, 2+ and 3+. Phosphopeptide identification results were accepted only if the Mascot scorespass statistically confidence (p < 0.05) and the identified sequence ranked as the top match. The search results in MASCOT were exported in eXtensive Markup Language data (.XML) format.The raw data were converted into files of mzXML format. Peptide identification results from each LC-MS/MS run and the corresponding XML files were used for quantitative analysis by IDEAL-Q as described previously
Wu JL, Wu HY, Tsai DY, Chiang MF, Chen YJ, Gao S, Lin CC, Lin CH, Khoo KH, Chen YJ, Lin KI. Temporal regulation of Lsp1 O-GlcNAcylation and phosphorylation during apoptosis of activated B cells. Nat Commun. 2016 Aug 24;7:12526 PubMed: 27555448