Phosphorylation events within
cancer cells often become dysregulated, leading to oncogenic signaling and abnormal cell growth.
Phosphopeptides derived from aberrantly phosphorylated
proteins that are presented on
tumors and not on normal tissues by
human leukocyte antigen (HLA) class I molecules are promising candidates for future
cancer immunotherapies, because they are
tumor specific and have been shown to elicit cytotoxic T cell responses. Robust
phosphopeptide enrichments that are suitable for low input amounts must be developed to characterize HLA-associated
phosphopeptides from clinical samples that are limited by material availability. We present two complementary mass spectrometry-compatible,
iron(III)-immobilized
metal affinity chromatography (
IMAC) methods that use either
nitrilotriacetic acid (NTA) or
iminodiacetic acid (IDA) in-house-fabricated columns. We developed these protocols to enrich for subfemtomole-level
phosphopeptides from cell line and human tissue samples containing picograms of starting material, which is an order of magnitude less material than what is commonly used. In addition, we added a
peptide esterification step to increase
phosphopeptide specificity from these low-input samples. To date, hundreds of
phosphopeptides displayed on
melanoma,
ovarian cancer,
leukemia and
colorectal cancer have been identified using these highly selective
phosphopeptide enrichment protocols in combination with a program called 'CAD Neutral Loss Finder' that identifies all spectra containing the characteristic neutral loss of
phosphoric acid from phosphorylated
serine and
threonine residues. This methodology enables the identification of HLA-associated
phosphopeptides presented by human tissue samples containing as little as nanograms of
peptide material in 2 d.