Evaluation of Protein Preparation Methods for Quantitative Analysis of Pig Muscle Proteome

High-throughput shotgun proteomics is often hindered by the incompatibility of detergents with mass spectrometry (MS), making sample preparation a critical bottleneck for accuracy and robustness. This challenge is amplified in muscle proteomics, where the high dynamic range of protein abundance requires highly efficient and reproducible workflows to capture low-abundance proteins. To address this, we performed a systematic benchmarking of five preparation methods-stacking-gel (SG), tube-gel (TG), solid-phase extraction (SPE), filter-aided sample preparation (FASP), and suspension traps (S-TRAP)-using the sarcoplasmic fraction of pig muscle. The protein extracts obtained were subjected to label-free semi-quantitative proteomic analysis using high-performance nano-liquid chromatography coupled to tandem MS. Qualitative and quantitative results were compared using bioinformatics and biostatistics tools. Our study identified 530 proteins with significant variations across methods. S-TRAP provided the highest identification depth, capturing the broadest proteome coverage. Conversely, the TG method demonstrated superior quantitative reproducibility, essential for detecting subtle physiological changes. For translational research, such as meat quality science or muscle-related clinical models, our findings imply that S-TRAP is the preferred choice for discovery-phase proteomics (biomarker hunting), while TG or SG should be prioritized for high-precision validation studies.