Using long columns to quantify over 9200 unique protein groups from brain tissue in a single injection on an Orbitrap...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Advancements in LC-MS/MS bottom-up proteomics primarily focused on mass spectrometer enhancements.
• The Orbitrap Astral offers high scan rates and sensitivity but is prohibitively expensive for many labs.
• Liquid chromatography (LC) advancements for deeper proteomics have lagged since Multidimensional Protein Identification Technology (MudPIT) in 2001.

Purpose of the Study:

• To develop a cost-effective, labor-efficient, and reproducible method for deeper proteomics.
• To optimize LC conditions using longer columns on a more affordable mass spectrometer.
• To enable deep proteome profiling with a single injection, avoiding fractionation.

Main Methods:

• Evaluation of long trap (up to 40 cm) and analytical columns (up to 600 cm).
• Optimization of sample loading, gradient time, and analytical column particle size.
• Utilized a 30 cm trap column and 250 cm analytical column with optimized conditions.
• Employed a 24-hour gradient on an Orbitrap Exploris 480 mass spectrometer.

Main Results:

• Optimized LC conditions, particularly column length, are key for deeper proteomics.
• Quantified over 9200 unique protein groups from brain tissue in a single injection.
• Achieved deep proteome profiling using a cost-effective Orbitrap Exploris 480 mass spectrometer.

Conclusions:

• Longer LC columns are crucial for achieving deeper proteomics with cost-effective instruments.
• The developed fractionation-free LC method enables deep proteome quantification in a single injection.
• This approach enhances reproducibility and reduces labor intensity in bottom-up proteomics.