Proteomics analysis of an individual formalin-fixed paraffin-embedded tissue section using isobaric-tag amplification.

RATIONALE: The comprehensive analysis of formalin-fixed paraffin-embedded (FFPE) tissues is essential for retrospective clinical studies. However, detecting low-abundance proteins and obtaining proteome-scale data from FFPE samples pose analytical challenges in mass spectrometry-based proteomics. To overcome this challenge, our study focuses on implementing an isobaric labeling approach to improve the detection of low-abundance target proteins in FFPE tissues, thereby enhancing the qualitative and quantitative analysis. METHODS: We employed an isobaric labeling approach utilizing synthetic peptides or proteins to enable the qualitative and quantitative measurement of target proteins in FFPE tissue samples. To achieve this, we incorporated tandem mass tag (TMT)-labeled recombinant proteins or synthetic peptides into TMT-labeled metastatic breast cancer FFPE tissues. Through this strategy, we successfully detect coexisting CD276 (B7-H3) and CD147 proteins while identifying over 6000 proteins using targeted analysis of individual FFPE tissue sections. RESULTS: Our findings provide compelling evidence that the incorporation of isobaric labeling, along with the inclusion of TMT-labeled peptides or proteins, greatly enhances the detection of target proteins in FFPE tissue samples. By employing this approach, we were able to obtain robust qualitative measurements of CD276 and CD147 proteins, showcasing its effectiveness in identifying more than 6000 proteins in FFPE samples. CONCLUSIONS: The integration of an isobaric labeling approach, in conjunction with synthetic peptides or proteins, presents a valuable strategy for enhancing the detection and validation of target proteins in FFPE tissue analysis. This technique holds immense potential in retrospective clinical studies, as it enables comprehensive analysis of low-abundance proteins and facilitating proteome-scale investigations in FFPE samples. By leveraging this methodology, researchers can unlock new insights into disease mechanisms and advance our understanding of complex biological processes.