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

The advantages of top-down proteomics (TDP) in the characterization of proteoforms, resulting from genetic variations, alternative splicing, and post-translational modifications (PTMs), have been well documented. However, TDP applications on limited samples have been less explored, and no direct comparison with the bottom-up proteomics (BUP) approach for the same scarce amounts of samples has been conducted to date. In this work, we processed ∼100-1000 HeLa cells using bottom-up and top-down workflows and subjected sample volumes equivalent to ∼25 and up to ∼250 HeLa cells to liquid chromatography-mass spectrometry (LC-MS)-based TDP and BUP analyses. Porous layer open-tubular (PLOT) columns were used for the separation of intact proteins in TDP MS, while traditional bead-packed columns were used for the BUP workflow. Up to 500 proteoforms and nearly 1300 proteins from cell lysates equivalent to ∼25 HeLa cells were identified in TDP and BUP, respectively. Interestingly, among all the unambiguously identified proteins from both ∼25 HeLa and ∼250 HeLa cell lysates in TDP, ∼20-30% were not identified in BUP under the same sample loading, suggesting significant complementarity between TDP and BUP approaches. Additionally, biologically relevant PTMs (e.g., acetylation, phosphorylation, and methylation) were reliably characterized in TDP as different proteoforms. We anticipate that TDP, enhanced by ultralow-flow PLOT chromatography columns coupled to MS, could be a supplementary or an alternative approach for limited-sample analysis, as it eliminates the need for protein digestion and minimizes sample cleanup, enabling rapid sample preparation while preserving proteoform information.