Full Mass Range ΦSDM Orbitrap Mass Spectrometry for DIA Proteome Analysis.

Optimizing data-independent acquisition methods for proteomics applications often requires balancing spectral resolution and acquisition speed. Here, we describe a real-time full mass range implementation of the phase-constrained spectrum deconvolution method (ΦSDM) for Orbitrap mass spectrometry that increases mass resolving power without increasing scan time. Comparing its performance to the standard enhanced Fourier transformation signal processing revealed that the increased resolving power of ΦSDM is beneficial in areas of high peptide density and comes with a greater ability to resolve low-abundance signals. In a standard 2 h analysis of a 200 ng HeLa digest, this resulted in an increase of 16% in the number of quantified peptides. As the acquisition speed becomes even more important when using fast chromatographic gradients, we further applied ΦSDM methods to a range of shorter gradient lengths (21, 12, and 5 min). While ΦSDM improved identification rates and spectral quality in all tested gradients, it proved particularly advantageous for the 5 min gradient. Here, the number of identified protein groups and peptides increased by >15% in comparison to enhanced Fourier transformation processing. In conclusion, ΦSDM is an alternative signal processing algorithm for processing Orbitrap data that can improve spectral quality and benefit quantitative accuracy in typical proteomics experiments, especially when using short gradients.

Parallelized Acquisition of Orbitrap and Astral Analyzers Enables High-Throughput Quantitative Analysis.

The growing trend toward high-throughput proteomics demands rapid liquid chromatography-mass spectrometry (LC-MS) cycles that limit the available time to gather the large numbers of MS/MS fragmentation spectra required for identification. Orbitrap analyzers scale performance with acquisition time and necessarily sacrifice sensitivity and resolving power to deliver higher acquisition rates. We developed a new mass spectrometer that combines a mass-resolving quadrupole, the Orbitrap, and the novel Asymmetric Track Lossless (Astral) analyzer. The new hybrid instrument enables faster acquisition of high-resolution accurate mass (HRAM) MS/MS spectra compared with state-of-the-art mass spectrometers. Accordingly, new proteomics methods were developed that leverage the strengths of each HRAM analyzer, whereby the Orbitrap analyzer performs full scans with a high dynamic range and resolution, synchronized with the Astral analyzer's acquisition of fast and sensitive HRAM MS/MS scans. Substantial improvements are demonstrated over previous methods using current state-of-the-art mass spectrometers.

Phase-Constrained Spectrum Deconvolution for Fourier Transform Mass Spectrometry.

This Article introduces a new computationally efficient noise-tolerant signal processing method, referred to as phased spectrum deconvolution method (ΦSDM), designed for Fourier transform mass spectrometry (FT MS). ΦSDM produces interference-free mass spectra with resolution beyond the Fourier transform (FT) uncertainty limit. With a presumption that the oscillation phases are preserved, the method deconvolves an observed FT spectrum into a distribution of harmonic components bound to a fixed frequency grid, which is several times finer than that of FT. The approach shows stability under noisy conditions, and the noise levels in the resulting spectra are lower than those of the original FT spectra. Although requiring more computational power than standard FT algorithms, ΦSDM runs in a quasilinear time. The method was tested on both synthetic and experimental data, and consistently demonstrated performance superior to the FT-based methodologies, be it across the entire mass range or on a selected mass window of interest. ΦSDM promises substantial improvements in the spectral quality and the speed of FT MS instruments. It might also be beneficial for other spectroscopy approaches which require harmonic analysis for data processing.

Experimental strategies to improve drug-target identification in mass spectrometry-based thermal stability assays.

Mass spectrometry (MS)-based thermal stability assays have recently emerged as one of the most promising solutions for the identification of protein-ligand interactions. Here, we have investigated eight combinations of several recently introduced MS-based advancements, including the Phase-Constrained Spectral Deconvolution Method, Field Asymmetric Ion Mobility Spectrometry, and the implementation of a carrier sample as improved MS-based acquisition approaches for thermal stability assays (iMAATSA). We used intact Jurkat cells treated with a commercially available MEK inhibitor, followed by heat treatment, to prepare a set of unfractionated isobarically-labeled proof-of-concept samples to compare the performance of eight different iMAATSAs. Finally, the best-performing iMAATSA was compared to a conventional approach and evaluated in a fractionation experiment. Improvements of up to 82% and 86% were demonstrated in protein identifications and high-quality melting curves, respectively, over the conventional approach in the proof-of-concept study, while an approximately 12% improvement in melting curve comparisons was achieved in the fractionation experiment.