Full Mass Range PhiSDM Orbitrap Mass Spectrometry for DIA Proteome Analysis

Optimizing data-independent acquisition (DIA) methods for proteomics applications often requires balancing spectral resolution and acquisition speed. Here we describe the full mass range application of Phase-constrained Spectrum Deconvolution Method (PhiSDM) for OrbitrapTM mass spectrometry in real time, and compare its performance to the standard enhanced Fourier transformation (eFT). We show that the increased resolving power of PhiSDM is beneficial in areas of high spectral complexity and comes with a greater ability to resolve low-abundance signals. In a 2-hour analysis of 200 ng HeLa digest, this resulted in an increase of 8% and 16% in the number of quantified protein groups and peptides, respectively. As the acquisition speed becomes even more important when using short chromatographic gradients, we further applied PhiSDM methods to a range of shorter gradient lengths (21, 12, and 5 min). While PhiSDM improved identification rates and spectral quality in all tested gradients, it proved particularly advantageous for the 5 min gradient. Here the number of protein groups and peptides identified increased by >15% in comparison to eFT processing. In conclusion, PhiSDM is an alternative signal processing algorithm for processing FTMS data that can increase spectral quality and provide quantification benefits for LC-MS acquisitions especially when using short gradient.