Top-down mass spectrometry (TDMS) offers the theoretical possibility of capturing the full molecular complexity of every gene product (proteoform) present in a cell. However, characterizing proteoforms in high-throughput proteomic experiments remains challenging. Electrospray ionization generates wide charge state distributions for each proteoform, leading to reduced spectral dynamic range. Furthermore, large cations are difficult to transfer to high resolution mass analyzers without undesired fragmentation. As a consequence, only the most abundant proteoforms are typically detected and selected for fragmentation, and the effect dramatically worsen for proteins > 30 kDa. We performed extensive TDMS characterization of the budding yeast proteome, to characterize subcellular compartment-specific proteoforms using an Orbitrap Eclipse Tribrid mass spectrometer. We first benchmarked the TDMS performance using LC-separated standard proteins from 8.5-29 kDa (Sigma Aldrich). Results indicate that reduced Orbitrap analyzer pressure and optimized ion transmission produce a 2-4 fold increase in spectral signal-to-noise ratio (SNR) in comparison to previous Orbitrap Tribrid MS. This SNR gain leads to more accurate on-the-fly charge state determination and facilitates m/z to mass deconvolution. Importantly, the improved performance in intact mass determination (i.e., MS1) at high resolving power (120,000 at 200 m/z or higher) is reached without compromising the quality of data-dependent (DDA) fragmentation spectra (i.e., MS2) obtained via higher-energy collisional dissociation. To characterize large (30-60 kDa) proteoforms, we applied a targeted data acquisition strategy: a quadrupole-isolated small m/z region (1.5 m/z wide) was first expanded via Proton Transfer Charge Reduction to enable accurate determination of proteoforms’ average mass (at medium resolution, 15’000 at 200 m/z) and subsequently fragmented via collisional dissociation. Results obtained indicate that the targeted PTR-based data acquisition method doubles the number of protein entry and proteoform identifications (at 1% FDR) compared to DDA experiments, allowing the characterization of large proteoforms fundamental for yeast central metabolism.