Background: Due to the high complexity and wide dynamic range of the human proteome, several predicted protein products have not yet been identified in proteomic experiments. Proteins that have been predicted but are currently unverified are classified as ‘missing proteins’ (MP). These proteins could provide essential clues to the interpretation of biological processes, and offer new research and therapeutic strategies to address the remaining clinical challenges.
Method: Proteins were extracted and digested from fresh frozen melanoma metastases samples 1. Peptides were TMT-labelled, fractionated and analyzed by nLC-MS/MS on a Q Exactive HF-X mass spectrometer. Raw data were processed with Proteome Discoverer v2.3. Protein evidence (PE) was determined using the criteria from neXtProt and the C_HPP 2. The bioinformatics analysis of relational networks between proteins that correlated with novel PE5 proteins was performed by ingenuity pathway analysis. Protein family annotation (PFAM) of the PE2 proteins was detected using DAVID.
Results: In this study, nine MPs were confidently identified by mass spectrometry in melanoma metastases. Some MPs significantly correlated with proteins that possess identical PFAM structural domains and other MPs were significantly-associated with cancer-related proteins. All proteins were classified based on the PE categories reported by neXtProt and annotated according to the HUPO guidelines 3. Four proteins were uniquely identified within the context of metastatic cancer progression, although previously supported only by transcript presence, and five proteins previously marked as proteins of uncertain evidence and suspected to be pseudogenes were explicitly linked to mechanisms of melanoma metastasis. In addition, 24 other missing proteins were identified in up to 140 melanoma metastases.
Conclusion: Deep proteomic analyses were performed on more than 100 tissue samples from malignant melanoma patients. To our knowledge, this is the first study where unknown and novel proteins have been annotated in metastatic melanoma tumor tissue.