Immunopeptidomics With TIMS and PASEF

Contents 04.................................Unmask the immunopeptidome with timsTOF 05.................................A paradigm shift in mass spectrometry 06.................................MOMA: Address highly similar peptides in MHC analysis 07.................................TIMS and PASEF 08.................................TIMS enabled polygon filtering scan 12..................................Characteristics of peptides presented by MHC class I or II 14..................................Advancing immunpeptidomics with quantitative mass spectrometry 16..................................Dissecting the powerhouse: PASEF and TIMS 17..................................Benefit from the unique polygon filtering and PASEF 20.................................Driving separation with PepSep HPLC columns 21.................................Nano-flow sensitivity made easy and robust 22.................................Breakthrough with Bruker timsTOF Ultra 24.................................Master immunopeptidomics with Bruker ProteoScape and BPS Novor 26.................................From bench to bedside — translating novel immunotherapeutic strategies 29.................................Further readings and references04 Low abundance targets? Unmask the immunopeptidome with timsTOF Traditional methods stumble Hear from the expert Nicola Ternette, Ph.D., Associate Professor, Antigen Discovery, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom ▪ Invisible targets: Low-abundance MHC peptides hide, masked by overwhelming noise, hindering discovery ▪ Quantity concerns: Limited sample tissue? Existing methods struggle to see the full picture, missing crucial immune targets ▪ Twin troubles: Isobaric peptides? Traditional tools get confused by lookalikes. This is the reason we need improved selectivity and ion mobility, to discover hidden antigens ▪ Singly charged species: MHC class I immunopeptides typically consist of 8-14 amino acids. They frequently occur as singly charged species due to their nature ▪ Complex data analysis: Immunopeptidomics generates large amounts of complex data that are nontrivial to analyze and interpret. It is particularly challenging to address the largely increased search space when the source of peptides is unknown. This necessitates sophisticated bioinformatic tools, including de novo search algorithms, to identify MHC-bound peptides and potentially discover new immunotherapy targets WATCH PODCAST05 ▪ Exclusive sensitivity: timsTOF's unique separation power reveals hidden low-abundance peptides ▪ Less tissue, more data: Even precious, scant samples unlock rich immunopeptidomic insights ▪ Sharper focus:Accurate targeting of MHC peptide properties makes sure no ions are missed A paradigm shift in mass spectrometry ▪ No more isobaric shadows: TIMS combined with advanced data analysis unveil hidden gems, separating true hits from imposters ▪ Precision single charge targeting: Unique polygon filtering only includes singly charged peptides masses of interest, not unwanted noise ▪ Bioinformatics powerhouse: Bruker ProteoScape™ with its integrated BPS Novor module unleashes the power of advanced de novo sequencing, rapidly deciphering unknown peptides (~2 minutes for 1-hour acquisition, >1000 spectra/second)Sequence RLPQLPEQ[+0.98] Mobility 0.88 Calc. m/z 491.27182417 40 40 60 0.9 0.8 mobility (1/Ko) 1.0 45 min. Sequence THLLQQEL Mobility 0.83 Calc. m/z 491.27182417 Extracted Ion Chromatogram m/z 491.2718 RT = 42.5 min Extracted Ion Mobilogram m/z 491.2718 RT = 42.5 min 06 ▪ Separate and quantify isobaric immunopeptides with ultra-accuracy ▪ Boost sensitivity for reliable detection of near-identical peptides ▪ Unravel complex MHC patterns hidden by sequence similarities Did you know? MOMA means accuracy: MOMA (Mobility Offset Mass Aligned) leverages the fourth dimension of separation to reduce chimeric spectra and isolates isobaric peptides, ensuring accurate peptide identification and quantification MOMA: Address highly similar peptides in MHC analysisIon accumulation and serial elution of seperated ion packages in parallel Fast switching, synchronized quadrupole Time-of-flight (TOF) analyzer at µsec speed Parallel accumulation TIMS scan Quadrupole Collision cell E Gas Ion seperation based on mobility collisional cross section (CCS) Space focusing of ions with similar mobilities (CCS) Time focusing of ions (accumulation) t E N2 N2 N2 t E Gas 07 Did you know? CCS molecular fingerprints: Measures a peptide's Collision Cross Section (CCS) with TIMS, like a molecular fingerprint, showing its size and shape. This can be used to gain insights into peptide conformation and aid peptide identification ▪ Unmatched sensitivity: Space focusing on ions amplifies signal, revealing even the faintest MHC signatures ▪ Precise precursor control: Target short, singly charged MHC Class I peptides with pinpoint accuracy, no longer masked by larger molecules ▪ Deeper MHC insights: Uncover hidden ligands and subtle peptide differences, unlocking a wealth of immunological information TIMS and PASEF Ultra sensitivity for immunopeptidesIon Intensity MS/MS scans Isolation filter polygons HLA class I peptide-tailored 56.4% 42% 3.5% 0.6 0.8 1.0 1.2 1.4 1.6 1.8 250 500 750 1000 1200 1500 56.4% 42% 35% 3.5% 250 500 750 1000 1200 MHC-tailored 0.6 0 1 2 3 4 x104 Int. 200 400 600 800 1000 1200 1400 m/z 1.0 1.2 1.4 Mobility 1/ko Charge a + 1 a + 2 a ≥ + 3 HLA class I peptide-tailored 59.7% 40.2% 2.1% 0.6 0.8 1.0 1.2 1.4 1.6 1.8 250 500 750 1000 1200 1500 59.7% 40.2% 21% 2.1% 08 TIMS enabled polygon filtering scan Immunopeptidomics polygon filtering: Exemplary heatmaps of ions of specific charge state distribution and masses are targeted.1Data visualisation for further bioinformatics analysis 99-99-99-99-88-73-72-62-99 GPALGRSFL Peptide HLA Peptides Peptides elution m/z P1 P2 P3 P4 Parallel accumulation TIMS scan E Gas 0910 “De novo searches have been a critical aspect in my labs research for many years...”11 “…the ability to have an algorithm that delivers results with accuracy and precision at the speed of acquisition means we can assess immunopeptidomics samples at scale and in real time. This has major implications in how fast my group can translate research findings into actionable information, and it pushes this workflow even closer to delivering clinical impact of these analyses on an unprecedented timescale.” Prof. Anthony W Purcell, Head of Immunoproteomics Laboratory, Monash University12 Characteristics of peptides presented by MHC class I or II Characteristics MHC class I MHC class II Location Cell surface of nearly all nucleated cells including infected or abnormal cells (e.g. cancer cells) Cell surface of antigen-presenting cells, such as dendritic cells and macrophages Peptide source Intracellular proteins processed within the cytosol by the proteasome Extracellular proteins that are endocytosed by professional antigen-presenting cells and processed via the endosome and lysosome Binding groove Closed, more compact, typically more hydrophobic peptides Open, more flexible T-cell recognition Cytotoxic T-cells, triggering the elimination of infected/abnormal cells Helper T-cells, regulating the immune response and activating other immune cells Immune responses Eliminate infected or abnormal cells Regulate other immune cells Peptide length 8 to 14 amino acids 15 to 25 amino acids Online information bruker.com/immunopeptidomics Innovation with Integrity