DOI: 10.5061/dryad.4tmpg4fh7
title: LC-MS/MS data for WT human PANX1 with or without co-expressing Src-Y529F mutant

description: 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—are nonspecific and should not be used to interpret
 results related to PANX1 phosphorylation. We further provide evidence that
 neither tyrosine residue is a major phosphorylation site for Src kinase in
 heterologous expression systems. We call on the field to re-examine the
 existing paradigm of tyrosine phosphorylation-dependent activation of the
 PANX1 channel.
description: 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), Acetyl (N-term), Pyro-Glu (N-term Q),
 Deamidation (N/Q); Mass values: Monoisotopic; Peptide Mass Tolerance: 10
 ppm; Fragment Mass Tolerance: 0.02 Da; Max Missed Cleavages: 2. Mascot DAT
 files were parsed into Scaffold (Proteome Software) for validation,
 filtering and to create a non-redundant list per sample. Data were
 filtered using 1% protein and peptide FDR and requiring at least two
 unique peptides per protein.
description: # 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.