Protocol for endolysosomal proteomics in goat atrial tissue using a refined density-gradient approach

Here, we present a protocol to analyze endolysosomal (EL) proteins in goat atrial fibrillation (AF) tissue using a modified density-gradient, proteomic-based fractionation. This method separates organelles from tissue lysate, isolating sarcoplasmic reticulum, mitochondria (1.3 g/mL), and EL (1.04 g/mL) with minimal contamination. The protocol includes tissue isolation, homogenization, density-gradient processing, peptide analysis, data analysis, and protein network identification using discontinuous Percoll and sucrose gradients; it supports molecular and proteomic studies. For complete details on the use and execution of this protocol, please refer to Ayagama et al.¹ and Ayagama et al.².