Identification of Tyrosine O Sulfated Proteins in Cow Retina and the 661W Cell Line

Abstract

Lack of tyrosine O Sulfation compromises both rod and cone electroretinographic responses emphasizing the importance of this post-translational modification for vision. To identify tyrosine sulfated proteins in retina, cow retinal lysates were subjected to immunoaffinity purification using an anti-sulfotyrosine antibody. The tyrosine sulfated proteins were eluted from the column using a sulfotyrosine pentapeptide and identified using mass spectrometry. Similarly, tyrosine sulfated proteins secreted by the 661W cell line were identified. Proteins identified were vitronectin, fibronectin, fibulin 2, nidogen, collagen V alpha 2, complement component 3 and C4 and fibrinogen beta. All proteins were subjected to analysis by 'Sulfinator' to determine potential sulfated tyrosines.

Sulfoproteomics Workflow with Precursor Ion Accurate Mass Shift Analysis Reveals Novel Tyrosine Sulfoproteins in the Golgi

“…These challenges have resulted in sulfotyrosine underrepresentation in PTM databases and a dearth of site information, the latter of which has limited recognition of its potential role in crosstalk with tyrosine phosphorylation competing for the same site. To date, only a small number of sulfoproteins (<200 across all species) has been identified, based on a combination of western blotting and MS-based protein identification. , The latter approach does not directly identify sites of sulfation; however, software tools such as Sulfinator, PredSulSite , and Position-Specific-Scoring-Matrix can predict sulfation sites within identified proteins based on the conserved sequence preference in known sulfoproteins. However, such sites need to be experimentally verified, and this approach has, to our knowledge, not been applied to proteomic scale analysis.…”