Top-Down versus Bottom-Up Proteomics in Highly Sensitive LC-MS-Based Profiling of Limited Samples

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Top-down proteomics (TDP) excels at characterizing proteoforms, including those from genetic variations, alternative splicing, and post-translational modifications (PTMs).
• TDP applications for limited sample sizes remain underexplored, lacking direct comparisons with bottom-up proteomics (BUP).

Purpose of the Study:

• To compare TDP and BUP for analyzing scarce samples (∼25-250 HeLa cells).
• To evaluate the utility of ultralow-flow porous layer open-tubular (PLOT) chromatography in TDP for limited samples.

Main Methods:

• Liquid chromatography-mass spectrometry (LC-MS)-based TDP and BUP analyses were performed on ∼100-1000 HeLa cells.
• Porous layer open-tubular (PLOT) columns were used for TDP, while traditional bead-packed columns were used for BUP.
• Sample volumes equivalent to ∼25 and up to ∼250 HeLa cells were analyzed.

Main Results:

• TDP identified up to 500 proteoforms, while BUP identified nearly 1300 proteins from ∼25 HeLa cells.
• 20-30% of unambiguously identified proteins in TDP were not detected by BUP at similar sample loadings, indicating complementarity.
• Biologically relevant PTMs were reliably characterized as distinct proteoforms using TDP.

Conclusions:

• TDP, especially with ultralow-flow PLOT chromatography, is a powerful supplementary or alternative approach for limited-sample proteomic analysis.
• TDP offers rapid sample preparation by eliminating protein digestion and minimizing cleanup, while preserving intact proteoform information.