In-depth mining of the immunopeptidome of an acute myeloid leukemia cell line using complementary ligand enrichment and data acquisition strategies

Pandey, K. Priyabrat; Mifsud, Nicole A.; Sian, Terry C.C. Lim Kam; Ayala, Rochelle; Ternette, Nicola; Ramarathinam, Sri H.; Purcell, Anthony W.

doi:10.1016/j.molimm.2020.04.008

Cited by 17 publications

(24 citation statements)

References 57 publications

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“…The remaining imperfectness in the overlaps implies that the parallel use of different isolation methods can still boost the number of identified peptides strongly (Figure 1A, Supplementary Figure S2A). 52 The moderate differences in the MHC allotype distribution of peptides obtained by MAE and MHC-IAC are in agreement with the hypothesis that the antibodies applied in MHC-IAC might possibly introduce a bias toward certain MHC allotypes. 9 However, we demonstrated that antibody independent variations in the MHC-IAC workflow, namely, the manner of performing ultrafiltration, can shift the MHC allotype distribution of observed peptides similarly strong as MHC-IAC does in comparison to MAE.…”

Section: ■ Discussionsupporting

confidence: 83%

Mild Acid Elution and MHC Immunoaffinity Chromatography Reveal Similar Albeit Not Identical Profiles of the HLA Class I Immunopeptidome

et al. 2020

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To understand and treat immunology-related diseases, a comprehensive, unbiased characterization of major histocompatibility complex (MHC) peptide ligands is of key importance. Preceding the analysis by mass spectrometry, MHC class I peptide ligands are typically isolated by MHC immunoaffinity chromatography (MHC-IAC) and less often by mild acid elution (MAE). MAE may provide a cheap alternative to MHC-IAC for suspension cells but has been hampered by the high number of contaminating, MHCunrelated peptides. Here, we optimized MAE, yielding MHC peptide ligand purities of more than 80%. When compared with MHC-IAC, obtained peptides were similar in numbers, identities, and to a large extent intensities, while the percentage of cysteinylated peptides was 5 times higher in MAE. The latter benefitted the discovery of MHC-allotype-specific, distinct cysteinylation frequencies at individual positions of MHC peptide ligands. MAE revealed many MHC ligands with unmodified, N-terminal cysteine residues which get lost in MHC-IAC workflows. The results support the idea that MAE might be particularly valuable for the high-confidence analysis of post-translational modifications by avoiding the exposure of the investigated peptides to enzymes and reactive molecules in the cell lysate. Our improved and carefully documented MAE workflow represents a high-quality, cost-effective alternative to MHC-IAC for suspension cells.

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Section: ■ Discussionsupporting

confidence: 83%

Mild Acid Elution and MHC Immunoaffinity Chromatography Reveal Similar Albeit Not Identical Profiles of the HLA Class I Immunopeptidome

et al. 2020

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“…Synthetic peptides for the selected sequences were ordered from Mimotopes as pepsets with ∼80% purity. Pepsets were reconstituted in 0.1% formic acid (FA), pooled together, and spiked with a mixture of 11 iRT peptides to aid retention time alignment ( 42 ) before LC–MS/MS analysis using the same method as for the HLA peptide analysis ( 44 ). That is, peptides were loaded onto a PepMap Acclaim 100 C 18 trap column 5 μm particle size, 100 μm × 2 cm and 100 Å (Thermo Fisher Scientific) at 15 μl/min using an Ultimate 3000 RSLC nano-HPLC (Thermo Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

Immunopeptidogenomics: Harnessing RNA-Seq to Illuminate the Dark Immunopeptidome

Scull

Pandey

Ramarathinam

et al. 2021

Molecular & Cellular Proteomics

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This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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“…In accordance with these data, we note that recent studies showed that different enrichment and desalting procedures alter allele-specific pMHC distributions measured by LC− MS. 33,34 The authors concluded that if the sample amount is not limiting, multiple complementary procedures might be used for deeper immunopeptidome surveys. However, we propose that when sample amounts are limiting, a single strategy that yields the deepest coverage would be most appropriate.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Automated Ligand Purification Platform Accelerates Immunopeptidome Analysis by Mass Spectrometry

et al. 2020

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Major histocompatibility complex (MHC)-presented peptides (pMHC) give insight into T cell immune responses, a critical step toward developing a new generation of targeted immunotherapies. Recent instrumentation advances have propelled mass spectrometry to being arguably the most robust technology for discovering and quantifying naturally presented pMHC from cells and tissues. However, sample preparation has remained a major limitation due to timeconsuming and labor-intensive workflows. We developed a highthroughput and automated platform with enhanced speed, sensitivity, and reproducibility relative to prior studies. This pipeline is capable of processing up to 96 samples in 6 h or less yielding high-quality pMHC mixtures ready for mass spectrometry. Here, we describe our efforts to optimize purification and mass spectrometer parameters, ultimately allowing us to identify as many as almost 5000 pMHC I and 7400 pMHC II from as little as 2.5 × 10 7 Raji cells each. We believe that this platform will facilitate and accelerate immunopeptidome profiling and benefit clinical research for immunotherapies.

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In-depth mining of the immunopeptidome of an acute myeloid leukemia cell line using complementary ligand enrichment and data acquisition strategies

Cited by 17 publications

References 57 publications

Mild Acid Elution and MHC Immunoaffinity Chromatography Reveal Similar Albeit Not Identical Profiles of the HLA Class I Immunopeptidome

Mild Acid Elution and MHC Immunoaffinity Chromatography Reveal Similar Albeit Not Identical Profiles of the HLA Class I Immunopeptidome

Immunopeptidogenomics: Harnessing RNA-Seq to Illuminate the Dark Immunopeptidome

Automated Ligand Purification Platform Accelerates Immunopeptidome Analysis by Mass Spectrometry

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