Protein Characterization by MALDI In-Source Decay Mass Spectrometry in Support of Safety Assessments of Genetically...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Genetics

Background:

• Mass spectrometry (MS) is crucial for characterizing transgenic proteins in genetically modified (GM) crops.
• Safety assessments of GM crops require high confidence in transgenic protein identity and structure.
• Existing methods may have limitations in comprehensive protein characterization, especially for complex proteins.

Purpose of the Study:

• To demonstrate the application of matrix-assisted laser desorption ionization in-source decay (ISD) mass spectrometry (MS) for transgenic protein analysis.
• To achieve high confidence in the sequences of plant-expressed transgenic proteins.
• To establish the biochemical equivalence of microbially produced protein surrogates for GM crop safety assessments.

Main Methods:

• Utilized matrix-assisted laser desorption ionization in-source decay (ISD) mass spectrometry (MS).
• Combined ISD with intact mass analysis and bottom-up proteomics.
• Employed top-down MS for identifying post-translational modifications and sequence verification.

Main Results:

• ISD confirmed 40-60 near-terminal residues, irrespective of protein size.
• Disulfide bond reduction/alkylation improved coverage for cysteine-rich proteins; negative ISD enhanced spectral quality for acidic proteins.
• Top-down MS identified post-translational modifications (e.g., truncations, N-terminal modifications) in plant-produced proteins.

Conclusions:

• The combination of top-down and bottom-up MS, particularly ISD, provides high confidence in transgenic protein sequencing.
• This analytical approach successfully establishes the biochemical equivalence between plant-produced proteins and microbial surrogates.
• The described MS strategy enhances the safety assessment of genetically modified crops.