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.
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Acknowledgements
This work was supported by the US National Institutes of Health with grants AI 033993 and GM O37537 (to D.F.H.).
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J.G.A., J.S. and D.F.H. designed the studies, and J.G.A. and P.D.T. executed them. S.A.P. and M.C. immunopurified HLA-associated peptides from colorectal cancer tumors that were resected by S.T.W. from consenting patients. A.M.P. and W.H.H. transfected FHIOSE cells with sHLA-A*0201 and isolated associated peptides. D.L.B. developed programs that were used during data analysis.
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Integrated supplementary information
Supplementary Figure 1 Full MS and MS2 Spectra demonstrating phosphopeptide sequencing using both CAD and ETD fragmentation.
A zoomed-in full MS spectrum showing the (M+3H)3+ peptide precursor ion isolated using a 3 m/z window for ETD and CAD fragmentation. The CAD MS2 spectrum demonstrates that the neutral loss of phosphoric acid from the precursor ion results in an MS2 spectrum that is difficult to interpret. The ETD MS2 spectrum of the same precursor enables the sequencing of this phosphopeptide because it preserves the labile post translational modification. Even though the CAD MS2 spectrum contains few sequence informative ions, it can be used to identify which precursors in the data are likely phosphopeptides because it contains the characteristic neutral loss of 98 Daltons. The “CAD Neutral Loss Finder” program is used to identify these spectra so they can be manually interpreted.
Supplementary Figure 2 Diagram of pressure bomb setup and microcapillary column fabrication.
Schematic depicting the pressure bomb setup used for handling fused silica microcapillary columns. The pressure cell is connected to a helium gas tank though a 3 way valve. Inside the pressure cell is a vial that contains a slurry of the desired packing material. A Kasil® 1624 fritted fused silica microcapillary column is inserted in the Teflon ferrule in the Swagelock brass fitting with the open end of the column in the packing slurry. The top of the pressure cell is screwed on, and the column is packed against the Kasil® 1624 frit when the inside chamber is pressurized with He gas.
Supplementary Figure 3 CAD MS2 spectra of a synthetic phosphopeptide compared to the same phosphopeptide found experimentally used for sequence validation.
Example MS and MS2 data are shown to illustrate our method of manual validation. A) Experimental data for the HLA-associated phosphopeptide RTLsHISEA. A lowercase “s” indicates that the serine is phosphorylated in the peptide sequence. The b- and y-ions for the esterified form of RTLsHISEA are shown with the expected mass shifts for esterification (14 Da) and phosphorylation (80 Da). A zoomed-in total ion current chromatogram (TIC) and a base peak chromatogram (Base Peak) from an enrichment experiment are displayed. Extracted ion chromatograms for the peptide precursor ion (561.2712 ± 5 ppm) and the MS2 of this precursor are also displayed. A zoomed-in Full MS spectrum depicts the (M+2H)2+ precursor ion at 561.2727 m/z, and the CAD MS2 spectrum of the precursor shows the relative abundances of the peptide fragment ions produced. B) An MS2 spectrum of the experimentally observed RTLsHISEA peptide labeled with the b- and y-ions produced from CAD fragmentation. Neutral losses of water from the b- and y-ions are labeled as “°” in the MS2 spectra. B-ions are labeled in blue, and y-ions are labeled in red. C) An MS2 spectrum of the synthetic RTLsHISEA peptide labeled with the b- and y-ions produced from CAD fragmentation. The synthetic peptide was used to validate that the correct sequence was assigned to the experimentally observed phosphopeptide.
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Abelin, J., Trantham, P., Penny, S. et al. Complementary IMAC enrichment methods for HLA-associated phosphopeptide identification by mass spectrometry. Nat Protoc 10, 1308–1318 (2015). https://doi.org/10.1038/nprot.2015.086
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DOI: https://doi.org/10.1038/nprot.2015.086
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