The quality and consistency in every sample preparation procedure is crucial for any scientific output. Therefore, it is of utmost importance to have easy, economic, and robust sample preparation protocols. Here, we describe a simple and robust bottom-up proteomic sample preparation strategy for identification and label-free quantification (LFQ) of proteins and phosphoproteins. The presented workflow is designed for large-scale application and involves easy scalable and well-known robust sample preparation techniques, such as cell lysis with SDS buffer under heat, protein precipitation using methanol/chloroform, tryptic digest, and commercially available TiO2 phosphopeptide enrichment kits. Over a sample set of 48 samples of only 200 mg fungal mycelium each, we quantified a median of 2937 proteins after processing in the IsoQuant software. The median peptide count was 10 peptides per protein leading to a median 65% sequence coverage. In addition, we identified a median of 3324 phosphopeptides (corresponding to 998 phosphoproteins) with 4874 phosphosites per sample. Over all samples, we achieved a median phosphopeptide enrichment efficiency of 77%. The distribution of serine/threonine/tyrosine (S/T/Y) phosphosites was 78.1%/21.2%/0.6%.
Keywords: Label-free quantification; Magnaporthe oryzae; Phosphopeptide enrichment; Phosphoproteomics; Proteomics; Sample preparation; TiO2.