MALDI-TOF Mass Spectrometry Profiling of Plasma Exosomes Evaluates Osteosarcoma Metastasis Zhenzhen Han
Identification of proteins from plasma-derived exosomes of from healthy controls and osteosarcoma patients by LC-MS/MS in label-free quantification. Lysis buffer (1% SDS, 8 M urea, 1x Protease Inhibitor Cocktail (Roche Ltd. Basel, Switzerland)) was added into the samples, vibrated and milling for 400 s for three times. The samples were then lysed on ice for 30 min and centrifuged at 15000 rpm for 15min at 4℃. The supernatant was collected and transferred to a new Eppendorf tube. The protein concentration was determined by using the BCA protein assay, and then 100 μg of protein per condition was transferred into a new Eppendorf tube and the final volume was adjusted to 100 μL with 8 M urea. 2 μL of 0.5 M TCEP was added and the sample was incubated at 37℃ for 1 h, and then 4 μL of 1 M iodoacetamide was added to the sample and the incubation was last for 40 minutes protected from light at room temperature. After that, five volumes of -20℃ pre-chilled acetone was added to precipitate the proteins overnight at -20℃. The precipitates were washed by pre-chilled 90% acetone aqueous solution for twice and then re-dissolved in 100 μL 100 mM TEAB. Sequence grade modified trypsin (Promega, Madison, WI) was added at the ratio of 1:50 (enzyme : protein, weight : weight) to digest the proteins at 37℃ overnight. The peptide mixture was desalted by C18 ZipTip, quantified by Pierce™ Quantitative Colorimetric Peptide Assay (23275) and then lyophilized by SpeedVac. Label free quantification was also performed using Peaks Studio based on results under 1% FDR. Relative abundance of peptide features(precursor peak area) was detected in multiple samples. Feature detection is performed separately on each sample, and then the features of the same peptide from different samples are reliably aligned together using a high-performance retention time alignment algorithm. Normalization was performed on total ion current (TIC) of the samples and normalized abundance is calculated from the raw abundance divided by the normalization factor. ANOVA was used for peptide and protein abundance calculation. Different expressed proteins were filtered if their fold change were over 1.5 and contained at least 2 unique peptides with significance over 13 (p<0.05).