Proteomics for cultivated meat: Evaluation of sample preparation methods and analytical workflows for label-free...

Cultivated meat production requires robust and validated analytical methods for comprehensive characterization. While transcriptomics-based approaches establish the foundational profile of molecular analysis, proteomics provides a direct estimate of the presence and abundance of proteins in cells and tissues, enhancing the scientific certainty in both product development and safety characterization. However, the industrial adoption of proteomics is currently hindered by technical complexity and a critical lack of analytical standardization, which leads to significant workflow-dependent variations in proteome coverage. To address this gap, we investigated the influence of key workflow steps (digestion, cleanup, LC-MS conditions) on the proteome profile of cultivated duck biomass. We compared five bottom-up sample preparation protocols - two traditional in-solution options (urea and SDC-based protocols), two device-based approaches (PreOmics iST and EasyPep kits), and an innovative protocol (SPEED), and demonstrated that device-based protocols offered the highest peptide yield and proteome coverage. However, optimization allowed cost-effective in-solution methods (SDC and SPEED) to achieve comparable performance. Specifically, an optimal digestion time of 3 h at 37 °C and the use of polymer-based desalting columns significantly enhanced protein identification (∼4500-5000 IDs). Moreover, data independent acquisition (DIA) provided deeper proteome coverage than data dependent acquisition (DDA) with higher precision (∼6500 vs 5000 IDs). The optimized and cost-effective sample prep protocols presented here establish a standardized framework for bulk bottom-up proteomics in cultivated meat.