contamination protein), where the amounts of exosome protein were still uncertain.
To solve this issue, we tested 10% of each tag-labeled sample using LC-MS/MS before mixing, and obtained the amount of exosome protein by a label-free method. High-abundance contamination proteins including all forms of IgG and FN were deleted from the list of identified proteins. The remaining proteins were assumed to be derived from exosomes. Then, all peptide intensities of exosome proteins were log2 transferred and their mean concentration was determined. The mean of log2 transferred peptide intensities reflected the relative amount of exosome proteins. The difference of means between samples of two time points was the log2 transferred ratio of their protein …show more content…
According to our experience, 1~2µg is the optimal amount for general nanoLC-MS/MS. However, we could only acquire 0.1~0.2µg exosome proteins from 4 mL of serum.
We designed an experiment, where we started with 36mL of standard serum. After the exosome extraction and protein digestion, we split up the sample into 9 aliquots (3 aliquots for sample A and 6 aliquots for sample B). For sample A, each aliquot of exosome protein was labeled with an iTRAQ tag 114, 115 and 116, respectively, and then mixed together. For sample B, each of 3 aliquots of exosome protein was labeled with iTRAQ-tags 114, 115 and 116, respectively, and the other 3 aliquots of exosome protein were labeled with an iTRAQ-tag 117. Sample A and sample B were analyzed separately by …show more content…
In order to investigate the importance of pro-migratory proteins in the exosomes, Panc-1 and MiaPaCa2 cells were exposed to serum derived from healthy controls or patients. While the serum of healthy controls had little impact on cell migration in a wound healing assay, faster wound closure was observed in cells exposed to serum derived from three out of four patients. Although the total change in migration potential was significantly higher in Panc-1 cells, a similar trend was observed in MiaPaCa2
To solve this issue, we tested 10% of each tag-labeled sample using LC-MS/MS before mixing, and obtained the amount of exosome protein by a label-free method. High-abundance contamination proteins including all forms of IgG and FN were deleted from the list of identified proteins. The remaining proteins were assumed to be derived from exosomes. Then, all peptide intensities of exosome proteins were log2 transferred and their mean concentration was determined. The mean of log2 transferred peptide intensities reflected the relative amount of exosome proteins. The difference of means between samples of two time points was the log2 transferred ratio of their protein …show more content…
According to our experience, 1~2µg is the optimal amount for general nanoLC-MS/MS. However, we could only acquire 0.1~0.2µg exosome proteins from 4 mL of serum.
We designed an experiment, where we started with 36mL of standard serum. After the exosome extraction and protein digestion, we split up the sample into 9 aliquots (3 aliquots for sample A and 6 aliquots for sample B). For sample A, each aliquot of exosome protein was labeled with an iTRAQ tag 114, 115 and 116, respectively, and then mixed together. For sample B, each of 3 aliquots of exosome protein was labeled with iTRAQ-tags 114, 115 and 116, respectively, and the other 3 aliquots of exosome protein were labeled with an iTRAQ-tag 117. Sample A and sample B were analyzed separately by …show more content…
In order to investigate the importance of pro-migratory proteins in the exosomes, Panc-1 and MiaPaCa2 cells were exposed to serum derived from healthy controls or patients. While the serum of healthy controls had little impact on cell migration in a wound healing assay, faster wound closure was observed in cells exposed to serum derived from three out of four patients. Although the total change in migration potential was significantly higher in Panc-1 cells, a similar trend was observed in MiaPaCa2