Bottom-up proteomics typically involves enzymatic digestion of proteins, generating a complex peptide mixture. These peptides are separated using reversed-phase ultrahigh-performance liquid chromatography (UHPLC) and analyzed using electrospray ionization (ESI) tandem mass spectrometry (MS/MS) in positive ion mode. Despite its widespread use, this approach has limitations, particularly in ionizing highly acidic or hydrophobic peptides and detecting certain post-translational modifications (PTMs). To overcome these challenges, alternative ionization methods, such as vacuum ultraviolet (VUV) atmospheric pressure photoionization (APPI), have been explored. In this study, we propose peptide analysis using a novel prototype APPI source employing soft X-ray photons. Soft X-ray photons possess orders of magnitude higher energy than VUV photons, enabling additional ionization pathways. Here, we present peptide ionization data using soft X-ray and VUV APPI in both positive and negative ion modes. Notably, soft X-ray photons exhibited a remarkable capacity to generate deprotonated peptides and hydrogen-deficient peptide radical anions ([M - 2H]), outperforming conventional VUV photons. Furthermore, collision-induced dissociation (CID) of [M - 2H] provided unique structural insight, facilitating PTM characterization. Our findings emphasize the significant potential of soft X-ray APPI in advancing peptide analysis and highlight the utility of negative ion mode for proteomic applications.