Global Proteome Survey -Transforming antibody-based affinity proteomics into a global discovery platform
Sammanfattning: Proteomics is expected to generate new insights into biological processes as well as identify novel biomarkers and therapeutic targets since most biological functions are transmitted through proteins. However, due to the complexity displayed by a proteome and inherent limitations associated with current methodologies, proteomic analyses often result in incomplete coverage and inconsistent measurements. Clearly, the development of novel high-performing proteomic platforms will be essential in order to successfully decipher the human proteome(s). This thesis, based on four original papers denoted I to IV, describes the development and applicability of a novel proteomic technology platform entitled Global Proteome Survey (GPS) capable of transforming affinity proteomics into a global discovery engine. The GPS methodology combines the best features of affinity proteomics and mass spectrometry, and is based on using antibodies specific for short C-terminal amino acid peptide motifs shared by many proteins. This opens up the possibility to identify and quantify significant portions of a proteome, while still using a limited set of binders in a specie independent manner (Paper I-IV). Furthermore, structural models were generated for a large set of the experimentally verified captured peptides and the matching antibodies (Paper III). The data generated novel insights into antibody-peptide interactions and showed that a few key residues were essential for establishing specificity and acting as anchor residues. The GPS assay reproducibility was extensively tested (Paper I, II, and IV) and displayed median coefficient of variations in the range of 10-20% in tissue profiling. In addition, the sensitivity was demonstrated by successfully targeting proteins present in a range of abundance values spanning over a million down to less than 50 copies per yeast cell (Paper I and II). In Paper IV, the first clinical proteomic application of GPS was demonstrated by generating in-depth proteomic insights of 52 breast tumor tissues. While using only 9 antibodies, the GPS-platform enabled identification and quantification of over 1300 proteins, and most importantly established a link between a molecular signature and tumor progression. Highly relevant and promising cancer-associated protein signatures related to histologic grade, estrogen receptor, and HER2/neu-status were identified. In conclusion, we have developed and demonstrated the quantitative capability, reproducibility, sensitivity, and coverage of the GPS methodology. It provides important methodological solutions to the complexity of proteome analysis and may act as a valuable tool for analyzing large numbers of clinical samples in an accurate, sensitive, and discovery-based manner. Hence, antibody-based affinity proteomics have been transformed into a global discovery platform and will pave the way for novel proteomic insights into complex molecular pathways in health and disease.
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