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.

• MeSH
• Atmospheric Pressure MeSH
• Photons MeSH
• Spectrometry, Mass, Electrospray Ionization * methods   MeSH
• Peptides * analysis   chemistry   MeSH
• Protein Processing, Post-Translational MeSH
• Proteomics methods   MeSH
• X-Rays MeSH
• Tandem Mass Spectrometry * methods   MeSH
• Ultraviolet Rays MeSH
• Chromatography, High Pressure Liquid methods   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Peptides * MeSH

A typical bottom-up proteomic workflow comprises sample digestion with trypsin, separation of the hydrolysate using reversed-phase HPLC, and detection of peptides via electrospray ionization (ESI) tandem mass spectrometry. Despite the advantages and wide usage of protein identification and quantification, the procedure has limitations. Some domains or parts of the proteins may remain inadequately described due to inefficient detection of certain peptides. This study presents an alternative approach based on sample acetylation and mass spectrometry with atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). These ionizations allowed for improved detection of acetylated peptides obtained via chymotrypsin or glutamyl peptidase I (Glu-C) digestion. APCI and APPI spectra of acetylated peptides often provided sequence information already at the full scan level, while fragmentation spectra of protonated molecules and sodium adducts were easy to interpret. As demonstrated for bovine serum albumin, acetylation improved proteomic analysis. Compared to ESI, gas-phase ionizations APCI and APPI made it possible to detect more peptides and provide better sequence coverages in most cases. Importantly, APCI and APPI detected many peptides which passed unnoticed in the ESI source. Therefore, analytical methods based on chymotrypsin or Glu-C digestion, acetylation, and APPI or APCI provide data complementary to classical bottom-up proteomics.

• Keywords
• acetylation, chemical ionization, photoionization, proteomics,
• MeSH
• Acetylation MeSH
• Atmospheric Pressure MeSH
• Chymotrypsin * MeSH
• Spectrometry, Mass, Electrospray Ionization methods   MeSH
• Peptides MeSH
• Proteomics * MeSH
• Chromatography, High Pressure Liquid methods   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chymotrypsin * MeSH
• Peptides MeSH