Yue Xuan1, 2, Yue Zhou3, Sebastien Gallien2, 4, Pedro Navarro1 , Oleksandr Boychenko5, Joshua Nicklay6, Jenny Ho7, Scott Peterman8, Ken Miller8
Purpose: A novel and streamlined high resolution MS1 based quantitation data-independent acquisition (HRMS1–DIA) workflow is developed aiming at proteomics large-cohort studies.
Methods: Experiments were performed on a capillary chromatographic separation with 1h gradient or nanoLC with 2h gradient online coupled to a high-resolution accurate mass (HRAM) mass spectrometer operated in an MS1-based quantitative data-independent acquisition mode.
Results: A 60-minute capillary LC- DIA MS method was developed with a flow rate of 1.2ul/min without compromising sensitivity along with high-throughput. The initial data demonstrate > 7000 proteins were reliably quantified. For a higher proteome coverage, a 120-minute DIA MS method based on the nano-LC separation is developed, where ~20% more proteins were quantified. In addition to the 1% FDR rate of DIA data analysis, a roll-up statistics strategy is applied to calculate the p-Value of each peptide, effectively improving quantitation precision > 90% by removing the interferences from the real quantitative signals. As a result, the ratios of the mixed three proteomes accurately reflect the expected values
1) Thermo Fisher Scientific, Bremen, Germany; 2) Thermo Fisher Scientific, Precision Medicine Research Center, Cambridge, MA, USA; 3) Thermo Fisher Scientific, Shanghai, China; 4) Thermo Fisher Scientific, Paris, France; 5) Thermo Fisher Scientific, Germering, Germany; 6) Thermo Fisher Scientific, New Jersey, USA; 7) Thermo Fisher Scientific, Hemel Hempstead, UK; 😎 Thermo Fisher Scientific, San Jose, USA