Increased throughput of proteomics analysis by multiplexing high-resolution tandem mass spectra

Area of Science:

• Analytical Chemistry
• Biochemistry
• Mass Spectrometry

Background:

• High-resolution Fourier transform mass spectrometry (FTMS) offers high specificity.
• The speed of tandem FTMS analysis is a limitation compared to lower-resolution instruments.

Purpose of the Study:

• To develop a faster, FTMS-based tandem mass spectrometry (MS/MS) analysis method.
• To improve the throughput of FTMS analysis for peptide identification.

Main Methods:

• A novel FTMS method was developed by accumulating MS/MS fragment ions from multiple precursors.
• The multiplexed spectrum was deconvoluted into individual MS/MS spectra post-acquisition.
• Peptide identification was performed using a search engine on concatenated spectra.

Main Results:

• The new FTMS method achieved a 2.5-3.0-fold increase in throughput compared to conventional FT MS/MS.
• The method was validated both computationally (in silico) and experimentally (in situ) on an LTQ Orbitrap mass spectrometer.
• Experimental results aligned with theoretical performance expectations.

Conclusions:

• The developed multiplexed FTMS method significantly boosts analytical speed.
• This approach overcomes the throughput limitations of conventional FT MS/MS, enhancing its competitiveness.
• The method offers a viable strategy for high-throughput peptide identification using FTMS.