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Fig. 1 | Clinical Proteomics

Fig. 1

From: Comparative analysis of differentially abundant proteins quantified by LC–MS/MS between flash frozen and laser microdissected OCT-embedded breast tumor samples

Fig. 1

Sample processing workflow. a Proteins were extracted from five invasive breast cancer specimens using three different methods, generating a total of 15 protein samples. In Method 1, flash-frozen specimens were homogenized (FF/HOM) and protein isolated using the illustra triplePrep kit. In Methods 2A and 2B, sections from OCT-embedded specimens were laser microdissected to isolate tumor cells only (OCT/LMD-T) or both tumor and stromal cells (OCT/LMD-TS), respectively. In both Methods 2A and 2B, the laser microdissected material was then incubated at 37 °C for 10 min followed by protein isolation using the illustra triplePrep kit. Extracted proteins were trypsin digested and TMT-labeled. TMT-labeled peptides were analyzed using 2D-LC–MS/MS and proteins quantified using Proteome Discoverer v1.4 followed by data analysis to identify differentially expressed proteins among the three different sample storage/processing methods. b Sample-to-TMT channel mapping. Samples were analyzed in 3 TMT sets with 7 channels in each set. In each TMT set, channel 126 contained the QCP (quality control pooled sample) while the remaining 6 channels contained the individual TMT-labeled samples. The last three TMT channels in set 3 contained technical replicates, one for each sample storage/preparation method

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