Assessment and refinement of sample preparation methods for deep and quantitative plant proteome profiling

Summary A major challenge in the field of proteomics is obtaining high quality peptides for comprehensive proteome profiling by liquid chromatography mass spectrometry for many organisms. Here we evaluate and modify a range of sample preparation methods using photosynthetically active Arabidopsis leaf tissues from several developmental timepoints. We find that inclusion of FASP-based on filter digestion improves all protein extraction methods tested. Ultimately, we show that a detergent-free urea-FASP approach enables deep and robust quantification of leaf proteomes. For example, from 4-day-old leaf tissue we profiled up to 11,690 proteins from a single sample replicate. This method should be broadly applicable to researchers working on difficult to process samples from a range of plant and non-plant organisms. Abbreviations Chloro Methanol/Chloroform Extraction FASP Filter Aided Sample Prep GO Gene Ontology IAA Iodoacetamide LFQ Label Free Quantification MS/MS Tandem mass spectrometry TF Transcription Factor UA Urea Extraction 1D 1 Dimensional 2D 2 Dimensional