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RESEARCH PRODUCT

Evaluation of FASP, SP3, and iST Protocols for Proteomic Sample Preparation in the Low Microgram Range

Toszka BohnUte DistlerStefan TenzerJörg KuharevMalte SielaffTobias BoppJennifer Hahlbrock

subject

Proteomics0301 basic medicineReproducibilityChromatography030102 biochemistry & molecular biologyChemistryMicrogramReproducibility of ResultsGeneral ChemistryCommercial kitProteomicsBiochemistrySpecimen HandlingWorkflow03 medical and health sciences030104 developmental biologySample SizeProteomeHumansSample preparationBottom-up proteomicsHeLa CellsTotal protein

description

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 μg range using varying amounts of HeLa cell lysate (1-20 μg of total protein). All three workflows showed similar performances for 20 μg of starting material. When handling sample sizes below 10 μg, 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 μg range. Even when digesting 1 μg 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 (R

yearjournalcountryeditionlanguage
2017-10-11Journal of Proteome Research
https://doi.org/10.1021/acs.jproteome.7b00433