Complementary IMAC enrichment methods for HLA-associated phosphopeptide identification by mass spectrometry

Area of Science:

• Biochemistry
• Immunology
• Oncology

Background:

• Dysregulated phosphorylation in cancer cells drives oncogenic signaling.
• Aberrantly phosphorylated proteins presented by HLA class I molecules on tumors are promising immunotherapy targets.
• Characterizing these phosphopeptides requires robust enrichment methods for limited clinical samples.

Purpose of the Study:

• To develop highly selective phosphopeptide enrichment protocols for low-input clinical samples.
• To enable the identification of human leukocyte antigen (HLA)-associated phosphopeptides for cancer immunotherapy.
• To improve the characterization of tumor-specific phosphopeptides from limited material.

Main Methods:

• Two complementary mass spectrometry-compatible iron(III)-immobilized metal affinity chromatography (IMAC) methods using nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) columns were developed.
• Protocols were optimized for subfemtomole-level phosphopeptide enrichment from picograms of starting material.
• A peptide esterification step was incorporated to enhance phosphopeptide specificity in low-input samples.

Main Results:

• The developed methods successfully enriched phosphopeptides from cell lines and human tissues with picograms of starting material, an order of magnitude less than commonly used amounts.
• Hundreds of phosphopeptides from melanoma, ovarian cancer, leukemia, and colorectal cancer were identified using these protocols combined with the 'CAD Neutral Loss Finder' program.
• The methodology allows for the identification of HLA-associated phosphopeptides from as little as nanograms of peptide material in 2 days.

Conclusions:

• The presented IMAC-based phosphopeptide enrichment strategies are highly selective and efficient for low-input clinical samples.
• This methodology significantly advances the identification of tumor-specific phosphopeptides for cancer immunotherapy development.
• The ability to analyze nanogram quantities of peptide material accelerates the discovery of novel cancer targets.