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The small size of ectosomes makes their isolation and obtaining the appropriate protein yield for liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic analyzes a considerable methodological challenge. Especially isolation of ectosomes from limited amount of body fluids of cancer patients means that a much smaller amount of protein is available for LC-MS/MS. The SP3 (solid-phase-enhanced sample preparation) method used by us so far [1] works when it is possible to obtain the appropriate amount of ectosomal protein as a result of scaling cell cultures. The aim of this research was to develop a method of sample preparation for LC-MS/MS based of S-Trap microcolumn technique that would give the same quality results despite using less protein.
Two cell lines were used in these research: anaplastic thyroid carcinoma (8305C) and normal thyroid follicular (Nthy-ori 3-1) cells. Ectosomes were isolated from conditioned media concentrated by low-vacuum filtration by differential centrifugation, and prepared for LC-MS/MS using SP3 or S-Trap techniques. Then LC-MS/MS was used to analyze the protein content of the ectosome proteome. Next, Gene Ontology (GO) analysis was performed using UniProt Database to classify identified proteins according to the biological processes, molecular functions and their cellular origin.
Using the SP3 method we identified 410 proteins in 8305C ectosomes and 558 proteins in Nthy-ori 3-1 ectosomes. S-Trap technique increased the numbers of identified proteins to 915 in 8305C ectosomes and to 804 proteins in Nthy-ori 3-1 ectosomes. For 8305C and Nthy-ori 3-1 ectosomes, 304 and 357 proteins were identified by both protocols , respectively. Alongside the proteins identified regardless of chosen sample preparation method (SP3 or S-Trap) provided significant number of protein that were not identified by the other one. According to GO the most abundant groups of proteins for both types of ectosomes were those connected with cytosolic or membrane origin. In 8305C ectosomes, several cancer-associated proteins were found, which suggests their possible role in cancer promotion.
Acknowledgements: The research was funded by the BioS Priority Research Area under the program “Excellence Initiative –Research University” at the Jagiellonian University in Krakow (U1U/P03/DO/13.17).
[1] Surman et al., , Int J Mol Sci. 2020, 21(8): 2934.
