Evaluation of high dynamic range MS1 acquisition methods on a hybrid Orbitrap mass spectrometer

Advancements in MS-based proteomics have enabled the analysis of the proteome comprehensively and quickly. However, accurately identifying and quantifying low-abundant human proteins remains difficult due to the wide range of protein abundance found in tissues and body fluids. Gas phase fractionation methods like BoxCar as well as BoxCar Assisted MS Fragmentation (BAMF) have addressed this challenge by adding multiple narrow mass-to-charge segments to fill the Orbitrap more efficiently. In our study, we have further developed these ideas. We made modifications to an Orbitrap^TM Exploris 480 mass spectrometer to allow gas phase fractionation and intelligent MS1 multiplexing schemes with optimized injection times for high dynamic range Orbitrap MS1 scans.

Pierce HeLa samples were analyzed by LC-MS/MS with 60min-per-run methods. The analysis was performed on a Thermo Scientific^TM Vanquish^TM Neo UHPLC system with a Thermo Scientific^TM PepMap^TM column (2μm, 150μm x 15cm) connected to a Thermo Scientific^TM Orbitrap^TM Exploris 480 mass spectrometer. The instrument was operated in DDA mode with Orbitrap-MS1 resolution at 120,000, and ddMS2 scans acquired using 2-Th quadrupole isolation windows and a resolution of 15,000. The raw data was evaluated with Proteome Discoverer^TM using the CHIMERYS^TM search engine.

We have performed data acquisition with proof-of-concept MS acquisition methods to achieve higher dynamic range (HDR) in Orbitrap based MS1 scans. This approach encompasses the on-the-fly decomposition of MS1 spectra into two full mass range sub[CC2] -scans, which are reconstituted as one afterwards. For gas phase fractionation of the ion current, we have implemented two different strategies, an equidistant mode (HDR equidistant) and a dynamic mode (HDR dynamic) to fractionate horizontally along the m/z range. Both methods profit from using low fill times for fractions with high-abundant signals and high fill times for fractions with low-abundant signals, achieving high dynamic range across the fully assembled MS1 spectrum. Gas phase fractionation for HDR leads to a significant increase of intra spectrum MS1 complexity when a tryptic HeLa digest is applied. Both approaches result in a substantial increase of the signal-to-noise ratio of Orbitrap MS1 scans with gain factors >20 for lower abundant ions. Enabling HDR also leads to a significant increase of global peptide isotopic patterns found in a 1h LCMS run of a HeLa proteolytic digest. The HDR equidistant mode results in detection of additional 48% isotopic patterns while additional 59% are detected in dynamic mode when compared to a control. Our findings confirm that the HDR approaches works in principle, leaving room for further improvements on identification and quantification of proteins with a wide range of abundance.