Identification of 7 000-9 000 Proteins from Cell Lines and Tissues by Single-Shot Microflow LC-MS/MS

A current trend in proteomics is to acquire data in a "single-shot" by LC-MS/MS because it simplifies workflows and promises better throughput and quantitative accuracy than schemes that involve extensive sample fractionation. However, single-shot approaches can suffer from limited proteome coverage when performed by data dependent acquisition (ssDDA) on nanoflow LC systems. For applications where sample quantities are not scarce, this study shows that high proteome coverage can be obtained using a microflow LC-MS/MS system operating a 1 mm i.d. × 150 mm column, at a flow-rate of 50 μL/min and coupled to an Orbitrap HF-X mass spectrometer. The results demonstrate the identification of ∼9 000 proteins from 50 μg of protein digest from Arabidopsis roots, 7 500 from mouse thymus, and 7 300 from human breast cancer cells in 3 h of analysis time in a single run. The dynamic range of protein quantification measured by the iBAQ approach spanned 5 orders of magnitude and replicate analysis showed that the median coefficient of variation was below 20%. Together, this study shows that ssDDA by μLC-MS/MS is a robust method for comprehensive and large-scale proteome analysis and which may be further extended to more rapid chromatography and data independent acquisition approaches in the future.̀.