Evaluation of Fast and Sensitive Proteome Profiling of FF and FFPE Kidney Patient Tissues

The application of proteomics to fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) human tissues is an important development spurred on by requests from stakeholder groups in clinical fields. One objective is to complement current diagnostic methods with new specific molecular information. An important goal is to achieve adequate and consistent protein recovery across and within large-scale studies. Here, we describe development of several protocols incorporating mass spectrometry compatible detergents, including Rapigest, PPS, and ProteaseMax. Methods were applied on 4 and 15 mu m thick FF tissues, and 4 mu m thick FFPE tissues. We evaluated sensitivity and repeatability of the methods and found that the protocol containing Rapigest enabled detection of 630 proteins from FF tissue of 1 mm(2) and 15 mu m thick, whereas 498 and 297 proteins were detected with the protocols containing ProteaseMax and PPS, respectively. Surprisingly, PPS-containing buffer showed good extraction of the proteins from 4 mu m thick FFPE tissue with the average of 270 protein identifications (1 mm(2)), similar to the results on 4 mu m thick FF. Moreover, we found that temperature increases during incubation with urea on 4 mu m thick FF tissue revealed a decrease in the number of identified proteins and increase in the number of the carbamylated peptides.Proteomic

Evaluation of Fast and Sensitive Proteome Profiling of FF and FFPE Kidney Patient Tissues

The application of proteomics to fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) human tissues is an important development spurred on by requests from stakeholder groups in clinical fields. One objective is to complement current diagnostic methods with new specific molecular information. An important goal is to achieve adequate and consistent protein recovery across and within large-scale studies. Here, we describe development of several protocols incorporating mass spectrometry compatible detergents, including Rapigest, PPS, and ProteaseMax. Methods were applied on 4 and 15 μm thick FF tissues, and 4 μm thick FFPE tissues. We evaluated sensitivity and repeatability of the methods and found that the protocol containing Rapigest enabled detection of 630 proteins from FF tissue of 1 mm(2) and 15 μm thick, whereas 498 and 297 proteins were detected with the protocols containing ProteaseMax and PPS, respectively. Surprisingly, PPS-containing buffer showed good extraction of the proteins from 4 μm thick FFPE tissue with the average of 270 protein identifications (1 mm(2)), similar to the results on 4 μm thick FF. Moreover, we found that temperature increases during incubation with urea on 4 μm thick FF tissue revealed a decrease in the number of identified proteins and increase in the number of the carbamylated peptides