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Category:
Natural Sciences

Lecturer:
Dr. Jan Muntel, Senior Research Scientist, Biognosys AG, Schlieren, Switzerland

Place:
DKFZ, TP3 building, ground floor, seminar room B0.300, Im Neuenheimer Feld 580

Host:
GPCF TechTalk Series

Description:
Comprehensive, reproducible and precise analysis of large sample cohorts is one of the key objectives of quantitative proteomics. Data-independent acquisition (DIA) methods developed to powerful workflow to achieve this goal. Instead of selecting precursors for fragmentation and downstream peptide identification, the whole mass range is sequentially scanned; potentially enabling comprehensive coverage of the entire sample. For benchmarking of the workflow, a HeLa sample was analyzed by different gradients and compared to the widely used data-dependent acquisition (DDA) method. Over all tested gradients, DIA resulted in twice as many peptide identifications and for short gradient it resulted even in more identifications than theoretically possible with the DDA approach (on a Q Exactive HF mass spectrometer). A mixing experiment of different proteomes showed also a better precision and accuracy of DIA-based quantification compared to DDA. The potential of DIA for cancer research was then tested in a small lung cancer study based on formalin-fixed paraffin-embedded (FFPE) tissue. Within only 2.5h instrument time, 5,500 proteins per sample were quantified with low CVs (~10%). Statistical analysis of the data revealed large changes within the cancer proteome (2,000 proteins in significantly altered abundance). An interesting finding was a suppression of the complement system through the C1QBP repressor which has been previously reported as mechanism of the tumor to evade the host cell response. In this talk, the basics and data analysis of DIA mass spectrometry will covered including spectral library based and new hybrid approaches omitting the spectral library generation step. The potential of DIA methods in cancer research will be demonstrated with small lung cancer studies based on FFPE and fresh frozen tissue.

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