Posttranslational Modifications Of Connexin26 Identified By MALDI-TOF/TOF Mass Spectrometry

Abstract

Gap junctions play important roles in auditory function. Mutations in the Cx26 gene are the predominant cause of inherited nonsyndromic deafness. Some Cx26 deafness mutations directly disrupt the intercellular molecular and/or ionic signaling pathway, while others may affect the location and character of posttranslational modifications (PTMs) governing channel assembly, function and biological regulation. Mass spectrometry was used to determine if Cx26 PTMs occur at sites of deafness-causing mutations. Cx26 was isolated from HeLa cells at >95% purity by immunoaffinity followed by metal chelate chromatography using carboxyl-terminal hexahistidine and hemagglutinin tags. In-gel and in-solution enzymatic digestions were carried out in parallel with trypsin, chymotrypsin and endoproteinase-GluC. Peptides were recovered with and fractionated from reverse-phased C8 beads by stepwise elution with increasing concentrations of organic solvent. Using an ABI4800 MALDI-TOF/TOF-MS, spectra were acquired from each elution step, thereby improving detection of low abundance peptides in complex mixtures and maximizing sequence coverage. Acquisition, processing and interpretation parameters were further optimized to improve ionization and fragmentation of hydrophobic connexin peptides. MALDI-TOF-MS and MALDI-TOF-MS/MS sequence coverage values obtained were significantly above those reported for other mammalian membrane proteins. Total Cx26 sequence coverage by MALDI-TOF-MS was 75.2%, with 31.1% sequence confirmed by MALDI-TOF-MS/MS. Improved ionization and sequencing of Cx26 peptides (especially transmembrane pore-lining domains) were further achieved with a Waters nano-liquid chromatography-coupled electrospray ionization quadrupole-TOF-MS. Several different PTMs of Cx26 were identified, many of which were at sites of deafness-causing mutation. The PTMs included phosphorylation, acetylation, methylation, citrullination, hydroxylation, γ-carboxylation and palmitoylation. Knowledge of the location and character of Cx26 PTMs will be instrumental in guiding experiments to understand how cellular mechanisms of channel regulation can become altered and lead to losses in auditory function. Supported by GM36044, DC7470, NS56509 (ALH) & NS046593 (HL).