LC-MS/MS data for WT human PANX1 with or without co-expressing Src-Y529F mutant

Protein phosphorylation is one of the major molecular mechanisms regulating protein activity and function throughout the cell. Pannexin 1 (PANX1) is a large-pore channel permeable to ATP and other cellular metabolites. Its tyrosine phosphorylation and subsequent activation have been found to play critical roles in diverse cellular conditions, including neuronal cell death, acute inflammation, and smooth muscle contraction. Specifically, the non-receptor kinase Src has been reported to phosphorylate Tyr198 and Tyr308 of mouse PANX1 (equivalent to Tyr199 and Tyr309 of human PANX1), resulting in channel opening and ATP release. Although the Src-dependent PANX1 activation mechanism has been widely discussed in the literature, independent validation of the tyrosine phosphorylation of PANX1 has been lacking. Here, we show that commercially available antibodies against the two phosphorylation sites mentioned above—which were used to identify endogenous PANX1 phosphorylation at these two sites—..., Purified hPANX1 protein with/without co-expressing mSrc Y529F mutant was resolved in SDS-PAGE gel and the band corresponding to hPANX1 protein was cut and subjected to in-gel digestion with trypsin. Half of each digested sample was analyzed by nano LC-MS/MS with a Waters M-Class HPLC system interfaced with a ThermoFisher Fusion Lumos mass spectrometer. Peptides were loaded on a trapping column and eluted over a 75µm analytical column at 350nL/min; both columns were packed with Luna C18 resin (Phenomenex). The mass spectrometer was operated in data-dependent mode, with the Orbitrap operating at 60,000 FWHM and 15,000 FWHM for MS and MS/MS respectively. The instrument was run with a 3 s cycle for MS and MS/MS.
Data were searched using a local copy of Mascot (Matrix Science) with the following parameters. Enzyme: Trypsin/P; Database: SwissProt Human (concatenated forward and reverse plus common contaminants); Fixed modification: Carbamidomethyl (C) Variable modifications: Oxidation (M), Ac..., , # LC-MS/MS data for WT human PANX1 without co-expressing Src-Y529F mutant

[https://doi.org/10.5061/dryad.4tmpg4fh7](https://doi.org/10.5061/dryad.4tmpg4fh7)

The raw data of LC-MS/MS experiment on WT human PANX1 with [MSB56652A.raw] or without [MSB56651A.raw] co-expressing Src-Y529F mutant in HEK293T cells.

The data has been acquired on a Fusion Lumos mass spectrometer (Thermo Scientific) using the Xcalibur software. Raw data files can be processed using MS convert utilities in ProteoWizard, which is freely available at [https://proteowizard.sourceforge.io/download.html](https://proteowizard.sourceforge.io/download.html). The result file can be viewed with the Scaffold free viewer, which can be downloaded from proteomesoftware.com.