Considering Measurement Time and Depth in diaPASEF Plasma Proteomics

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Mass spectrometry (MS)-based plasma proteomics is vital for clinical research.
• Data-independent acquisition (DIA) with ion mobility (diaPASEF) enables high-throughput proteomic analysis.
• The optimal spectral library generation for DIA proteomics remains debated.

Purpose of the Study:

• To evaluate spectral library generation methods for plasma proteomics on a timsTOF HT system.
• To assess the impact of measurement time and library depth on protein and peptide identification.
• To compare experimental versus in silico spectral libraries for high-throughput plasma proteomics.

Main Methods:

• Utilized data-independent acquisition (DIA) with ion mobility (diaPASEF) on a timsTOF HT system.
• Constructed and compared experimental and in silico spectral libraries.
• Analyzed the relationship between measurement time, library depth, and identification numbers.

Main Results:

• Increased measurement time for spectral libraries enhanced protein and peptide identifications.
• In silico libraries yielded lower proteomic depth compared to extensive experimental libraries.
• Experimental libraries increased peptide identifications by 14% over in silico libraries.

Conclusions:

• Experimental spectral libraries are superior for deep peptide identification in high-throughput plasma proteomics.
• The choice of spectral library significantly impacts proteomic depth and identification accuracy.
• Experimental libraries support the growing trend towards peptide-centric proteomic studies.