Thin-diaPASEF: diaPASEF for maximizing proteome coverage in single-shot proteomics.

Area of Science:

• Proteomics
• Mass Spectrometry
• Biomarker Discovery

Background:

• Proteomics using mass spectrometry (MS) provides deep insights into complex proteomes.
• The timsTOF MS platform with parallel accumulation-serial fragmentation (PASEF) offers high scan speeds and spectral quality.
• Bruker's timsTOF HT with TIMS-XR technology enhances dynamic range and analysis depth for high sample loadings.

Purpose of the Study:

• To validate and optimize data-independent acquisition (diaPASEF) parameters on the timsTOF HT.
• To develop an improved diaPASEF method for enhanced proteomic analysis.
• To establish a high-throughput proteomic analysis system for biomarker discovery.

Main Methods:

• Utilized Bruker's timsTOF HT mass spectrometry platform.
• Developed and validated a Thin-diaPASEF method by optimizing key parameters.
• Combined Thin-diaPASEF with an improved Lycopersicon esculentum lectin method for plasma analysis.

Main Results:

• Identified 9,400 proteins in a single shot from HEK cells with a false discovery rate <1%.
• Achieved the highest protein identification number for the timsTOF MS series in standard human cultured cells.
• Identified over 5,000 proteins in a 24-sample/day plasma analysis using Thin-diaPASEF and lectin enrichment.

Conclusions:

• The Thin-diaPASEF method offers high quantitative accuracy and consistency for deep proteomic analysis.
• The optimized system demonstrates high proteome coverage and suitability for biomarker discovery.
• This advancement pushes the boundaries of proteomic analysis on the timsTOF MS platform.