Evaluation of FASP, SP3, and iST Protocols for Proteomic Sample Preparation in the Low Microgram Range.
Efficient and reproducible sample preparation is a prerequisite for any robust and sensitive quantitative bottom up proteomics workflow. Here, we performed an independent comparison between single pot solid phase enhanced sample preparation (SP3) filter aided sample preparation (FASP) and a commercial kit based on the in StageTip (iST) method. We assessed their performance for the processing of proteomic samples in the low mg range using varying amounts of HeLa cell lysate (1 20 mg of total protein) All three workflows showed similar performances for 20 mg of starting material. When handling sample sizes below 10 mg, the number of identified proteins and peptides as well as the quantitative reproducibility and precision drastically dropped in case of FASP. In contrast, SP3 and iST provided high proteome coverage even in the low mg range. Even when digesting 1 mg of starting material, both methods still enabled the identification of over 3000 proteins and between 25 000 and 30 000 peptides. On average, the quantitative reproducibility between experimental replicates was slightly higher in case of SP3 (R2 0.97 (SP3) R2 0.93 (iST) Applying SP3 toward the characterization of the proteome of FACS sorted tumor associated macrophages in the B16 tumor model enabled the quantification of 2965 proteins and revealed a "mixed" M1/M2 phenotype.
Journal of proteome research