Min Huang, Xiujie Sun, and Yue Zhou
Purpose: To improve the proteome coverage and label-free quantitation performance of limited number of mammalian cells.
Methods: Different sample preparation methods and LC-MS parameters were compared for protein identification and quantitation performance evaluation. Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer was used for data acquisition. MS2 HCD ion trap scan was used for peptide identification and high resolution MS1 precursor was used for label free quantification. Data analysis was performed by Thermo Scientific™ Proteome Discoverer™ 2.4 with SequestHT and MSPepSearch.
Results: RapiGestlysis buffer, lower reaction volume and higher enzyme-protein ratio could achieve higher protein and peptide IDs (i.e., deeper proteome coverage). Around 2000 proteins and 6000 peptides were identified in 40 cells using the optimized method. And around 4300 proteins and 25000 peptides were identified in 800 cells. Median CVs of protein and peptide abundances were less than 20% and 11% in lower and higher loading amount samples, respectively, and the dynamic range could reach to 5 orders of magnitudes in both lower and higher loading amount samples.
Thermo Fisher Scientific, Shanghai, China