Selective Affinity Enrichment of Nitrotyrosine-Containing Peptides for Quantitative Analysis in Complex Samples

Area of Science:

• Biochemistry
• Proteomics
• Molecular Biology

Background:

• Protein tyrosine nitration is implicated in inflammatory and aging-related diseases.
• Mass spectrometry analysis of nitrotyrosine (NT) peptides is challenging due to low abundance and signal suppression by non-nitrated peptides.
• Validated methods for NT-peptide enrichment are currently lacking.

Purpose of the Study:

• To develop and validate an immunoaffinity enrichment method for NT-peptides.
• To improve the characterization of the nitrotyrosine proteome.
• To enable quantitative analysis of protein tyrosine nitration in complex biological samples.

Main Methods:

• Immunoaffinity enrichment of NT-peptides using a specific antibody.
• Evaluation of the method using nitrated protein standards and whole-cell lysates.
• Application of the method for quantitative analysis in mouse lung samples after poly(I:C) treatment.

Main Results:

• Identification of 1881 NT sites from a nitrated whole-cell extract.
• Demonstration of the method's validity and its significant advancement for nitrotyrosine proteome characterization.
• Quantification of 237 NT sites in mouse lungs, showcasing its utility in quantitative analysis.
• Observation of higher affinity for N-terminal NT-peptides, suggesting a need for pan-specific antibodies.

Conclusions:

• The developed immunoaffinity-MS method is effective for enriching NT-peptides.
• This approach significantly advances the characterization of the nitrotyrosine proteome.
• The method is suitable for quantitative analysis of protein nitrotyrosines in complex samples.
• Future development of pan-specific NT antibodies is needed for unbiased, proteome-wide analysis.