Updated publication reference for PubMed record(s): 27148881. Synchronous beating of the heart is dependent on the efficient functioning of the cardiac intercalated disc (ID). The ID is composed of a complex protein network enabling electrical continuity and chemical communication between individual cardiomyocytes. Off late a lot of studies have shed light on increasingly prevalent cardiac diseases involving the ID. Insufficient knowledge of the ID composition makes it difficult to study these disease mechanisms in more detail. Here, for the first time using a combination of membrane enrichment, in-depth proteomics analysis and bioinformatics strategies, we have discovered a defined set of new putative ID proteins. An ID enriched membrane fraction, which included the ID markers connexin-43 (Cx43) and n-cadherin, was prepared using differential centrifugation. We quantitatively evaluated 3455 proteins for their enrichment in the ID-enriched membrane fraction with respect to their levels in the counterpart soluble fraction. An intuitive data filtering approach was used to generate a final set of 97 high potential ID proteins. These included well-known markers such as Cx43 and n-cadherin, but also several interesting novel candidates. We selected 4 candidates (Flotillin-2 (flot2), Nexilin (nexn), Popeye-domain-containg-protein 2 (popdc2) and thioredoxin-related-transmembrane-protein 2 (tmx2)) and confirmed their co-localization with n-cadherin, and thus their enrichment in the ID of human, dog and rat heart sections and isolated cardiomyocytes