Figure 2.

Optimization of the iTRAQ method significantly improved the quantification of tumor-derived exosomal proteins. (a) Routine protein measuring method determined the amounts of total protein (exosome protein plus contamination protein), where the amounts of exosome protein were still uncertain. To compensate for this, (b) we used a label-free method to determine the exosome content of each tag-labeled sample before mixing. The x axis and y axis labels (a and b) represent sample labeled by four different iTRAQ mass tags (114, 115, 116, or 117) and relative protein amount of every sample, respectively. (c) Initially 36 mL of standard serum was used to extract exosomes and obtain protein. In sample A, each aliquot of exosome protein was labeled by tag 114, 115, and 116, respectively. In sample B, the situation was similar, except the other three aliquots of exosome protein were labeled tag 117. (d) An average of 502 proteins were identified in sample A and 495 proteins were quantified, while an average of 603 proteins were identified in sample B and 562 proteins were quantified. For both samples, the intensities of tags 114, 115, and 116 were identical; but 13.5% more proteins were quantified in the Sample B.