ausblenden:
Schlagwörter:
DATA-INDEPENDENT ACQUISITION; FILTER DIAGONALIZATION METHOD;
FOURIER-TRANSFORM; PERFORMANCE EVALUATION; SENSITIVITY; ACCURACY;
COVERAGEBiochemistry & Molecular Biology;
Zusammenfassung:
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 (ISDM) 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 ISDM 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 ISDM methods to a range of shorter gradient lengths (21, 12, and 5 min). While ISDM 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, ISDM 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.
