Peptide Analysis by Soft X-ray Atmospheric Pressure Photoionization Mass Spectrometry

Area of Science:

• Analytical Chemistry
• Biochemistry
• Mass Spectrometry

Background:

• Bottom-up proteomics commonly uses reversed-phase ultrahigh-performance liquid chromatography (UHPLC) and electrospray ionization (ESI) tandem mass spectrometry (MS/MS).
• Conventional methods face challenges with ionizing acidic/hydrophobic peptides and detecting post-translational modifications (PTMs).
• Vacuum ultraviolet (VUV) atmospheric pressure photoionization (APPI) has been explored as an alternative ionization technique.

Purpose of the Study:

• To investigate the use of a novel prototype APPI source utilizing soft X-ray photons for peptide analysis.
• To compare soft X-ray APPI with VUV APPI in both positive and negative ion modes.
• To evaluate the potential of soft X-ray APPI for improved peptide ionization and PTM characterization.

Main Methods:

• Peptide ionization using a novel soft X-ray APPI source.
• Comparison of ionization efficiency and pathways between soft X-ray and VUV APPI.
• Analysis of peptide fragmentation (collision-induced dissociation - CID) of deprotonated species ([M - 2H]•-) generated by soft X-ray APPI.

Main Results:

• Soft X-ray APPI demonstrated superior ionization capabilities compared to VUV APPI.
• Soft X-ray photons effectively generated deprotonated peptides and hydrogen-deficient peptide radical anions ([M - 2H]•-) in negative ion mode.
• CID of [M - 2H]•- yielded unique structural information, beneficial for PTM identification.

Conclusions:

• Soft X-ray APPI presents a promising advancement for peptide analysis in bottom-up proteomics.
• The use of negative ion mode with soft X-ray APPI enhances the detection and characterization of peptides and PTMs.
• This novel technique offers potential solutions to limitations associated with conventional proteomic analysis methods.