Sample Preparation for Quantitative Proteome and Phosphoproteome Profiling of Maize Tissues

Zea mays (maize) is a globally important cereal crop and a key system for studying plant development and stress responses. Proteome profiling and phosphoproteome profiling provide direct, quantitative readouts of protein abundance and phosphorylation states, which offer insights into aspects of regulation and cellular function that transcript-level measurements alone cannot provide. Robust and reproducible methods are essential for generating accurate and biologically relevant data in proteomics studies. The complexity of plant tissues, however, poses challenges for developing reliable sample preparation workflows. Here, we describe a detailed sample preparation protocol for quantitative proteome and phosphoproteome profiling in maize. The protocol encompasses protein extraction, filter-aided sample preparation (FASP), peptide desalting, tandem mass tag (TMT)-based labeling for quantitative multiplexing, and complementary TiO₂ and Fe-NTA enrichment steps, yielding peptides suitable for analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). This approach enables the quantitative profiling of protein abundance and phosphorylation dynamics in maize tissues.