Deep Bottom-up Proteomics Enabled by the Integration of Liquid-Phase Ion Trap

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Bottom-up proteomics requires advanced peptide separation for comprehensive analysis.
• Liquid phase ion traps (LPITs) were initially proposed for ion manipulation and improved detection sensitivity.

Purpose of the Study:

• To establish an LPIT-reversed phase liquid chromatography-tandem mass spectrometry (LPIT-RPLC-MS/MS) platform for deep bottom-up proteomics.
• To evaluate LPIT as a peptide fractionation method for enhanced proteome coverage.

Main Methods:

• Integration of LPIT with RPLC-MS/MS for peptide separation.
• Utilizing LPIT's ability to separate peptides based on effective charges and hydrodynamic radii.
• Analysis of HeLa cell proteomes.

Main Results:

• LPIT demonstrated robust and reproducible peptide fractionation with high sensitivity.
• The LPIT-RPLC-MS/MS platform significantly increased peptide and protein detection.
• Achieved an approximate 89.2% increase in peptide coverage and 50.3% increase in protein coverage for HeLa cells.

Conclusions:

• LPIT serves as an effective and orthogonal peptide fractionation method for deep bottom-up proteomics.
• The LPIT-based approach offers high efficiency and low cost.
• This method has potential for routine application in deep proteomic studies.