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

Abstract

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.

Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry

“…This implies for the high recovery and sensitivity of our method and importantly it avoids the requirement of reservation of dedicated samples for enrichments of phospho-peptides and additional laborious sample processing38. Nevertheless, such peptides may contain cancer-specific phosphorylation patterns and therefore potentially represent attractive targets for cancer immunotherapy3132.…”

Current tools for predicting cancer-specific T cell immunity

“…65 In general, these methods allow identification of T cell (neo-) MHC ligands, including those containing posttranslational modifications. 72,77 For identification of HLA eluted peptides MS/MS spectra are matched to theoretical spectra of peptide sequences in databases using search engines like Mascot 78 or MaxQuant. 79 Generation of customized databases from genomic and transcriptomic information 70 allowed identification of private peptides that are not present in reference protein sequence databases.…”

Minimal Information About an Immuno‐Peptidomics Experiment (MIAIPE)

“…For example, post‐translationally modified pMHC (e.g., phosphorylations,30 symmetric vs asymmetric demethylation,31 O‐GlcNAc vs O‐GalNAc32) may be directly identified by enabling variable modifications in the database searches. Therefore, all raw and processed data should be submitted to a public proteomic or immunopeptidomic repository (e.g., the resources included in the ProteomeXchange Consortium [http://www.proteomexchange.org/], such as PRIDE, MassIVE, jPOST, and iProX repositories,33 or the recently created SysteMHC Atlas immunopeptidomic data repository [https://systemhcatlas.org/]34) to enable better collaborations among researchers and data reuse by third parties, to advance the field more efficiently.…”