Revolutionize Proteoform Analysis With Advanced Multi-Stage Mass Spectrometry

timsOmniTM TIMS-MS An eXtreme leap in deep proteoform sequencing and advanced structural elucidation Innovation with Integrity Welcome to the timsOmniTM, a transformative instrument poised to revolutionize mass spectrometry in systems biology. Designed to push the boundaries of scientific discovery, the timsOmni achieves extraordinary sensitivity, advances deep proteoform sequencing and delivers unprecedented precision in structural elucidation. Discovering the intricacies of proteoform diversity and gaining new insights into the functional plasticity of proteins is now within reach. The timsOmni further enables detailed structural elucidation of potential mutagenic impurities, revealing how subtle chemical modifications can transform benign small molecules into carcinogenic threats, corroborating the superior versatility of this groundbreaking technology. The timsOmni embraces the power of top-down mass spectrometry by harnessing the complete spectrum of electron-based fragmentation schemes, underscoring the value of complementary dissociation techniques. Empowered by advanced MSn eXd and ion enrichment methods, this transformative platform detects labile post-translational modifications (PTMs), produces complementary fragment ions to resolve ambiguous spectra, and reconstructs complex molecular information with OmniScape™ software, delivering biology-relevant insights with unmatched depth and accuracy. An eXtreme leap for electron-based fragmentation techniques Loop 1 2 3 4 5 6 Q2 Q5 Q8 Loop 1 2 3 4 5 6 Q2 Q5 Q8 Electron source Q2 omni-directional accumulation with resonance excitation RCID and MSn isolation Q5 omni-directional accumulation with trapped eXd Q8 omni-directional MSn ion enrichment and optical access Omni-directional MSn eXd with ion enrichment Powered by proprietary omni-directional lossless transfer and ion enrichment, signals for even the lowest-abundance ions are dramatically amplified. Through precise modulation of selected ion packets, any ion, regardless of its intensity, can be selectively targeted for electron-based fragmentation. 1) Quad isolation and direct omniaccumulation of precursor ions in section Q2. 2) Resonance excitation Collision Induced Dissociation (RCID) and isolation of target fragments for further interrogation. 3) Signal amplification via ion enrichment of selected MS2 CID fragments in Q8. 4) Transfer of the accumulated MS2 CID ion population in section Q5. 5) MS3 trapped eXd fragmentation of the enriched ion population 6) Ejection of MS3 product ions to TOF. Experience unmatched MS and MSn sensitivity Designed for results Ion enrichment is applied for PTM characterization of protein complexes sprayed under native conditions using the new NEOS source. Inlet Ion funnel Transfer multipole T0 Dual TIMS analyzer Introducing the timsOmni Advanced structural elucidation and deep proteoform sequencing by MSn eXd coupled to the power of TIMS. • TIMS separation to determine conformational heterogeneity and CCS to reveal structure • Trapped eXd for optimal precursor utilization boosting fragment ion yield • Precise control of electron-based fragmentation for detailed molecular profiling • Omni-directional MSn and trapped eXd with ion enrichment for unmatched sensitivity • Utilization of all PASEF modes for bottom-up proteomics and multiomics Explore the conformational landscape Collision induced unfolding (CIU) experiments are performed in T0 upstream of the TIMS. TIMS separation followed by eXd of selected conformations provides insights into higher order structure of proteins. Quadrupole mass fi lter Omnitrap® Collision cell with AIP Electron source and optics Q5 section H2 N H N H N N H R2 OH R4 Cα a x y R3 z· b c R1 O O O O OA TOF analyzer Why AIP is a game changer A small detail with a big impact A hot cathode produces a high-density electron beam, guided by a lens system into section Q5 for high efficiency eXd reactions with trapped ions, increasing product ion yield compared to other soft fragmentation methods. Nomenclature for the primary fragment ions types observed for peptides and proteins in tandem mass spectrometry. In ECD, when a protein or peptide ion captures a free electron, bond cleavage occurs rapidly and locally at the N–Cα bond, producing primarily c/z fragments. This non-ergodic process preserves labile post translational modifi cations like phosphorylation, glycosylation, sulfonation, etc. In CID, ions are gradually heated through multiple collisions, spreading energy throughout the molecule (ergodic). This causes only the lowest-energy bonds to break, mainly producing b/y fragments with limited sequence coverage and often poor PTM characterization. Combinations of ECD and CID using MSn eXd can be used to further enhance protein sequence coverage through formation of a/x, b/y and c/z ions. The AIP addresses mass discrimination effects in transferring ions to the TOF and results in a much broader mass range of ions being observed in the mass spectrum. irradiation time (ms) intensity 10 0 40 80 120 160 200 100 1000 10000 100000 [M+8H]8+ y70_6+ [M+8H]7+∙ z17_2+ [M+8H]6+∙∙ c10_1+ electron energy, eV relative intensity, % ECD electron capture [M+8H]7+∙ / [M+8H]+ [M+8H]9+∙ / [M+8H]+ electron ionization hECD ~7 eV EID >15 eV ~35 eV 0 4 8 12 16 20 0 10 20 30 electron ionization electron energy (eV) charge state -n ... -2 -1 0 +1 +2 ... +n 1 10 100 electron capture ECD hECD EID niECD EDD EI EED Electron energy optimization Optimum electron energies are identified for electron capture and ionization of ubiquitin [M+8H]8+ ions by tracing the signal of the charge-reduced [M+8H]7+∙ and the chargeincreased [M+8H]9+∙ product ions respectively as a function of electron energy. Precise modulation of electron kinetic energy and reaction time Precisely tune electron energy to access diverse fragmentation regimes. Adjust eXd reaction times and explore uncharted electron energy levels for different classes of analytes to achieve optimal product ion coverage. eXd Landscape Access to different electron-based fragmentation regimes becomes available by fine tuning electron energy from 0.1eV to >100 eV ECD – Electron Capture Dissociation hECD – hot Electron Capture Dissociation EED – Electron Excitation Dissociation EID – Electron Induced Dissociation EI – Electron Ionization niECD – Negative-Ion ECD EDD – Electron Detachment Dissociation Trapped ion eXd In trapped ion eXd, precursor ions are confined and rapidly fragment upon electron irradiation. Adapting the trapping time enables it to boost the fragment yield and reach optimal precursor consumption (>90%). This unique capability differs from traditional in-line electron-based fragmentation techniques. [M+8H]9+∙ / [M+8H]+ electron ionization d reaction times and explore uncharted electron energy levels for different classes of analytes to achieve Access to different electron-based fragmentation regimes becomes available by fine tuning electron energy from 0.1eV to >100 eV – Electron Capture Dissociation – hot Electron Capture Dissociation – Electron Excitation Dissociation – Electron Induced Dissociation – Electron Detachment Dissociation Q2 Resonance Trapped eXd Enrich Q5 Q8 CCID CCID CCID MS RCID RCID RCID ECD ECD ECD EID EID EID PD PD PD MULTI-STAGE MSn ION ACTIVATION NETWORK MS2 MS3 MS4 CCID MS2 RCID ECD EID PD Let's break it down! CCID occurs by axial acceleration of ions into the collision cell RCID occurs by resonant excitation in section Q2 of the Omnitrap platform eXd occurs in section Q5, receiving variable energy electrons from an external hot cathode Signal amplification via ion enrichment and also optical access is provided in section Q8 Powerful multimodal and multi-stage MSn Online multimodal fragmentation Multimodal fragmentation relies on combining information from first-generation fragment ion types, produced by complementary ion dissociation methods, to drastically increase sequence coverage. CCID – Collision-cell Collision Induced Dissociation RCID – Resonant-excitation Collision Induced Dissociation ECD – Electron Capture Dissociation EID – Electron Induced Dissociation PD – Photo-dissociation Successive stages of ion activation enable fi rst- and second-generation fragments to be selected for subsequent analysis. Distinct dissociation techniques can be employed at each MSn stage, allowing for highly customizable workfl ows. These tailored MSn strategies are well-suited to explore a broad range of analytes with diverse structural and chemical characteristics. Ion enrichment provides unprecedented levels of sequence and structural interrogation through signal amplifi cation, leveraging the high-capacity trapping sections across the Omnitrap platform. CCID CID ION ACTIVATION NETWORK MS2 CCID RCID Online multimodal fragmentation Multimodal fragmentation relies on combining information from first-generation fragment ion types, produced by complementary ion dissociation methods, to drastically increase sequence coverage. – Collision-cell Collision Induced Protein A Protein B Protein C Protein D Target charge states m/z MS2 MS1 MS2 Scan Cycle MS1 Top-down proteomics with charge DDA Charge DDA (cDDA) enables on-the-fly charge-state deconvolution during LC–MS analysis, allowing precise targeting of coeluting proteoforms across a wide dynamic range and eliminates redundant fragmentation of highly abundant species. By dynamically shifting the isolation window to non-overlapping regions of the m/z spectrum, cDDA significantly reduces chimeric spectra. Key Features • Ultra-fast on-the-fly charge-state deconvolution with intelligent m/z selection criteria. • Precursor selection targets nonoverlapping charge states producing high fidelity MS2 data. • Dynamic precursor accumulation optimizes ion fill times in the high-capacity sections of the Omnitrap platform for enhanced signalto-noise MSn spectra. • Automatic control of trapped eXd reaction times based on precursor charge state and user-defined target count criteria deliver optimal reaction efficiency. Averaging multiple TOF spectra sequentially at kHz rep rate produces high signal-to-noise ratio mass spectra. Multiple sequential MS2 scans result in high-fidelity isotopic distributions of fragment ions cDDA is based on ultra-fast on-the-fly identification of charge-state envelopes corresponding to individual proteoforms. A quick fact cDDA is tailored to top-down proteomics and differs from a standard top “n” method applied in bottom-up by using a more sophisticated precursor selection algorithm. scans result in high-fidelity isotopic distributions of fragment ions 33% sequence coverage and 71% outside of intrachain disulfide bonds a b c x y z IdeS (~25 kDa) (~100 kDa) 3 4 5 6 7 Time [min] Target counts 4E7 MS2 2E7 1E7 MS1 TIC Fc/2 F(ab)2 200 400 600 800 1000 1200 1400 1600 Dynamic precursor accumulation for superior LC-MS/MS sensitivity Top-down and middle-down LC-MS approaches enable rapid analysis of recombinantly expressed biologics. Targeted methods are particularly advantageous when high specificity and maximum sensitivity are required for protein sequencing, whether in complex matrices or purified samples typical of biopharmaceutical workflows. To further advance such analytical strategies, dynamic precursor accumulation actively regulates ion flow in MS/MS scans to optimally fill and compress the ion cloud within the trapping sections of the Omnitrap platform. By optimizing ion statistics, the fidelity of isotopic distributions is preserved with sequence coverage and analytical confidence further enhanced. Think of it this way Ion fill time is controlled dynamically in MS2 scans. Trapped eXd reaction time is adjusted according to precursor intensity and charge state. Dynamic precursor accumulation can also be applied in cDDA. Albert Heck Professor of Chemistry and Pharmaceutical Sciences, Utrecht University and Scientific Director of the Netherlands Proteomics Center “ Proteomics will finally go Protein-Centric by using the timsOmni” ›› IgdE Fabs 1000 0 1 11000 12000 13000 m/[z=1] Intensity 14000 15000 16000 HC c, a fragments LC c, a fragments ECD EID Comprehensive ion sequence ladders are ideal for de novo sequencing and human plasma antibody repertoire profiling. CDR1, CDR2, CDR3, S-S bridges a c Protein centric ECD analysis of a therapeutic antibody under denaturing conditions reveals CDR3 sequences. Sequencing Antibody Complementarity-Determining Regions Analyzing and monitoring circulating antibody levels is critical for characterizing the progression of a disease, identifying patients with delayed symptom onset and predicting potential longterm immunity. • Complementarity-Determining Regions (CDRs) are the hypervariable loops within the variable domains of the light and heavy chains primarily responsible for selectivity and affinity towards a specific antigen. Comprehensive ion sequence ladders are essential to characterize the unique CDRs. • CDR sequencing refers to the process of identifying the exact amino acid sequences within these loops to confirm identity, assess potential modifications, or facilitate antibody engineering. The superior efficiency of trapped eXd ensures comprehensive coverage of the critical functional regions in antibodies. Julia Chamot-Rooke CNRS Senior Scientist, Head of the Mass Spectrometry for Biology Unit at Institut Pasteur Paris, France “ The timsOmni delivers unmatched sensitivity and sequencing power - key to decoding multispecific antibody metabolites.” ›› *Data collected by Lucile Kogey-Fuchs PhD Student CIFRE #2023/0657 between Sanofi & Institut Pasteur Plasma samples (collected at different time points after administration) Immuno-enrichment (magnetic beads) timsOmni and nanoElute 2 t1 t2 t3 MS data fi les Data processing OSc Deconvoluted mass spectra Analytical strategy for characterizing in vivo biotransformation of biotherapeutics 90 100 110 120 130 140 150 160 170 102.0 102.5 103.0 103.5 104.5 105.5 Mass (kDa) Mass (kDa) 104.0 105.0 4h 24h 72h Proteoforms 4 - 11 Zoom Proteoforms Zoom 2 & 3 X Lc1 Hc1 Hc2 Lc2 Proteoform 1(administered) 4 5 6 7 8 9 10 11 Monitoring in vivo biotransformation products for improving the effi cacy of biotherapeutics Multispecific antibodies are rapidly emerging as a leading class of biotherapeutics, capable of simultaneously binding to multiple target antigens. These innovative modalities offer novel mechanisms of action, with enhanced efficacy, reduced risk of resistance, and fewer side effects compared to traditional therapies. However, unlike monoclonal antibodies, multispecifics are more prone to degradation in vivo, requiring thorough metabolite characterization. Top-down and middle-down MS approaches are particularly well-suited for such detailed structural analysis, as bottom-up strategies often fall short in capturing the complete proteoform landscape. Compounding this challenge is the extremely low abundance of these metabolites in in vivo samples, which makes their analysis especially difficult. The introduction of the timsOmni marks a significant advancement, ushering in a new era for the comprehensive characterization of multispecific antibody modalities. Time-resolved monitoring of in vivo biotransformation products highlighting preferential clipping of the LC1 and HC1 chains, responsible for reducing biotherapeutic effi cacy. Detection of very low abundance biotransformation products, ranging from 3 to 30 nM, is essential for investigating the in vivo stability of next generation biotherapeutics. Ole N. Jensen Professor Biomedical Mass Spectrometry and Systems Biology, University of Southern Denmark Director, PRO-MS, Danish National Mass Spectrometry Platform for Functional Proteomics “ The timsOmni technology and OmniScape software already impacted our strategies for intact protein and proteoform analysis. Multimodal MS/MS fragmentation and MS3 affords very high amino acid sequence coverage and accurate localization of post-translational modifications in histones.” ›› for intact protein and proteoform analysis. Multimodal MS/MS fragmentation and MS affords very high amino acid sequence coverage and accurate localization of post-translational modifications in histones.” Decrypting histone modifi cations is key to understanding disease Histones are crucial for DNA packaging, with their post-translational modifications (PTMs) playing a significant role in regulating gene expression by modifying chromatin structure. Understanding these histone modifications is paving the way for insights into diseases such as cancer, neurodegenerative disorders, cardiovascular diseases, and metabolic conditions. The high variability of modification sites in histone structures demands advanced analytical tools for accurate and reliable characterization and this can be achieved using the state-of-the-art fragmentation capabilities of the timsOmni along with sophisticated algorithms in OmniScape software to enable highly confident positional PTM assignments. Multimodal eXd (EID, ECD, CID, and ECD/CID) on charge states 19+ and 20+ provides complete sequence coverage for unambiguous localization of PTMs: 100% of sequence coverage H3.1K14ac proteoform identified a b c x y z From complexity to clarity OmniScape consistently identifies H3.1K14ac as the top proteoform match. Electron-based fragmentation methods individually provide the highest sequence coverage (~70-80%), while CID yields ~40%. Abraham Oluwole Postdoctoral researcher, Carol Robinson lab, University of Oxford, UK “ The ion enrichment mode in the timsOmni will facilitate increased scrutiny of low abundant fragments by MSn to achieve complete characterization for deeper insights” ›› 1000 500 1773 b1_1+ 1774 1775 1000 1006.4838 1118.2390 1771.3683 2132.7231 2314.1373 7951 1500 2000 2500 m/z BamE BamC BamA BamAB BamADE BamACDE BamABCDE 500 0 5.0x103 1.0x104 1.5x104 2.0x104 2.5x104 1500 2000 2500 3000 4000 5000 m/z m/z 6000 7000 8000 9000 40x BamE [M+7H]7+ MS2 RCID MS3 RCID of b12, 2+ 10x ion enrichment b12_2+ 500 1000 1500 2000 Beyond native MS, deep sequencing and PTM characterization of protein complexes Understanding how membrane proteins assemble to function in health and disease state is critical for drug development. This necessitates multi-level analysis of protein oligomeric states, subunit interactions, posttranslational modifications, and their binding to ligands and inhibitors. We illustrate how the integration of native and top-down mass spectrometry, with MSn and ion enrichment, offer direct and detailed structural insights. The analytical power of the timsOmni is exemplified using the β-barrel assembly machinery (Bam), a heteropentametric protein complex that facilitates the insertion of β-barrel proteins into the outer membrane of Gramnegative bacteria. Expand your options The timsOmni open data format allows convenient data analysis of native MS1 spectra, here processed with ProSightTM Native software Subunits are released by isCID, then MS2 RCID of BamE produces abundant fragments carrying the lipid modification, with b12 being an ideal target for MS3 analysis The mass of b1 unambiguously defines the position of the lipid at the N-terminal cysteine Native MS spectrum of Bam membrane protein complex Glycans are critical for modulating protein structure and function, including effi cacy as well as immunogenicity/toxicity of biopharmaceuticals. Variations in glycan structures can signifi cantly impact a protein's function, making their precise characterization essential in biopharmaceutical development. High-resolution characterization of glycans is vital, and diffi cult to achieve with CID, which often disrupts their fragile bonds. In contrast, eXd preserves glycan linkages while fragmenting the peptide backbone, enabling detailed mapping of glycosylation sites and providing unmatched structural resolution for therapeutic development and quality control. MS2 ECD of the Fc/2 subunit of the NIST IgG produces informative fragment ladders, with high abundance c61 7+ and c61 6+ fragments yielding the expected position and composition of G1F. The position of G0F modification was likewise confirmed by MS2 ECD on the corresponding glycoform. Sequence Coverage 91% Precision redefined Human glycans are built exclusively from only nine monosaccharides. eXd can be used to identify the site of the glycosylation and subsequent MSn yields high resolution information on composition, topology, and structure. a b c y z High-resolution characterization of glycans is vital, and diffi cult to achieve with CID, which often d preserves glycan linkages while fragmenting the peptide backbone, enabling detailed mapping of glycosylation sites and providing unmatched IdeS + Reduction 2x LC 2x Fd 2x Fc/2 (a+1)34_3+ (a+1)61_7+ (a+1)73_8+ (c+1)104_11+ (c+1)55_5+ (z+1)110_10+ (z+1)160_16+ (z+1)43_4+ (z+1)77_7+ (z+1)88_8+ (z+2)99_9+ a72_8+ b82_9+ b83_9+ c43_4+ c55_5+ c61_7+ c73_8+ y55_5+ y76_7+ y87_8+ G1F N-glycosylated c61 7+ fragment 1220 0 100 200 Intensity 300 1222 1224 1226 1228 1230 1232 1234 1238 1242 m/z 1236 1240 1244 Precision mapping of antibody glycosylation using eXd Combining results from multimodal and MSn modes performed on different precursor charge states of an industrial protease increases the sequence coverage to 92.6% a b c y z Anders Michael Bernth Giessing Science Manager, Novonesis, Lyngby, Denmark “ We use intact protein mass analysis to ensure performance, stability, and consistency of our very diverse protein product portfolio. Introduction of the timsOmni, with its Swiss Army knife versatility, redefines intact mass and topdown analysis with the precision, speed, and confidence needed to provide definitive analytical support in development and production of industrial enzymes.” ›› Direct infusion ESI SampleStream 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 m/z Automate sample delivery, save time The SampleStreamTM platform (IPT) coupled to timsOmni runs through HyStar, enabling automated workflows with rapid online buffer exchange. Pure protein signals with enhanced s/n ratio can be targeted for MSn eXd. Advancing the Applied Proteomics fi eld with multimodal fragmentation and MSn Bacterial enzymes are transforming industrial processes with their exceptional effi ciency, specifi city, and cost-effective production. In food manufacturing, these enzymes enhance fermentation, improve texture and clarify products by breaking down starches, proteins, and pectins. In pharmaceutical manufacturing, high-effi ciency enzymes streamline drug synthesis, resulting in improved yields and higher product quality. Multimodal fragmentation and MSn workfl ows enable precise mapping of protein sequences and modifi cation sites on intact molecules, accelerating optimization in synthetic biology. m/z Valérie Gabelica Full Professor, Analytical Chemistry, University of Geneva “ The timsOmni is our new Swiss-army knife for characterizing the structure and modifications of noncoding DNA, RNA, and oligonucleotide therapeutics” ›› m/z 200 400 600 23 - 22 - 10 - 20 - 19 -•• 18 -••• 20 -• m/z 718 21 - 11 - 12 - 13 - 14 - 15 - 16 - 17 - 18 - 19 - 20 - 21 - MS1 MS2 EDnoD MS3 RCID IRMPD MS3 RCID MS3 radicals IRMPD 21- ion, quad mass fi lter isolation 21- • ion, Omni Q2 isolation 21- m/z 684 683.6 683.8 m/z 684.2 684.4 717.8 718.0 m/z 718.4 718.6 800 1000 m/z 1200 1400 1400 1800 600 400 400 600 600 800 800 m/z 1000 1000 1200 1200 1400 1400 700 m/z 800 900 Deep characterization of oligonucleotides in negative ion mode by MSn DNA 46-mer oligonucleotide DNA and RNA are vital for cellular homeostasis, driving protein translation and regulating genes through mechanisms like epigenetics and interfering RNAs, and are increasingly being applied as valuable drug modalities across a range of diseases. Oligonucleotide characterization requires alternative fragmentation techniques because there are a limited number of canonical nucleotides and yet a diverse range of possible modifications. Mapping endogenous RNA modifications is key to understanding biological processes and topdown mass spectrometry with timsOmni is ideally positioned for precise sequencing. MSn eXd enables EDD and is particularly effective for intact top-down characterization of oligonucleotidebased therapeutics. Exploring uncharted opportunities Leading researchers are also applying photodissociation in conjunction with EDD for deeper structural characterization of molecules and leveraging exotic fragmentation schemes 83% sequence coverage in MS3 CID Data analysis performed in OligoQuestTM software Leading researchers are also applying photodissociation in conjunction with EDD for deeper structural characterization of molecules and leveraging exotic fragmentation schemes 83% sequence coverage in MS3 CID Data analysis performed in OligoQuestTM software Gustaf Hulthe Pharmaceutical Technology & Development, AstraZeneca R&D, Gothenburg, Sweden “ In many cases MS2 CID or MS2 EID alone cannot resolve the structure of observed impurities. So far, the timsOmni in MSn mode has solved the structural problems we have challenged it with, and without the need for synthesis. The versatile possibilities offered by this new platform may become a game changer.” ›› CI 6%* 2% C· CI N+ H2 6% 0.8% N N+ N 2% 2% 0.4% 23% 100% 4% C· CI HN+ 7% 3% CI N N C+ N N HN+ *Bond Cleavage leading to Cl localization * CI S S O O N OH o N N o CI N N N OH o o s N HN+ CI CI E N N N N+ N N N N s o s N N+ N+ CI o s N N N Three most probable positions of chlorine MS2 EID MS2 RCID The position of chlorine can only be ascertained by performing MS3 EID on MS2 CID product ions *Relative intensity Advanced Structural Elucidation of Small Molecules with MSn eXd In pharmaceutical development, detailed information on chemical structures is crucial to assess toxicity of impurities often produced during degradation or synthesis. New analytical workflows providing detailed structural elucidation eliminate the need to synthesize impurity standards, considerably accelerating drug development. Where is the chlorine? Aromatic chlorines are not reactive and are found in many drugs, while benzylic chlorines are potent alkylating reagents for DNA and proteins, rendering them carcinogenic. Locating the position of chlorination in trace impurities is critical as benzylic chlorines must be strictly controlled to minimize mutagenicity. The timsOmni goes beyond currently available MSn CID and MS2 EID activation methods, addressing such questions by accessing previously unreachable molecular bonds with MSn eXd. MS2 CID and MS2 EID can only eliminate one out of three possible chlorine positions. Gustaf Hulthe Pharmaceutical Technology & Development, AstraZeneca R&D, Gothenburg, Sweden “ In many cases MS resolve the structure of observed impurities. So far, the timsOmni in MS problems we have challenged it with, and without the need for synthesis. The versatile possibilities offered by this new platform may become a game changer.” ›› N+ H2 CI CI HN+ *Relative intensity unknown sequence de novo confi rmation FGTRAEWVIQ TFLERRNMA FFDEPRA CHANGAG.... Sequence tags partially known sequence PTM screening fragment matching >8k proteoforms MS-BLAST Search Scored protein Sequences Sequence maps Sequence maps Charge state plots OmniScape software a new era of confi dence in top-down sequence analysis OmniWaveTM for deisotoping highly complex mass spectra Advanced scoring system based on mass accuracy and isotope patterns Confirmation workflow applied to partially known protein sequences De novo algorithm for database search of top-down mass spectra PTM Screening for ultra fast proteoform identification of vast search spaces Innovation behind the solution The OmniWave algorithm calculates theoretical isotopic distributions for all possible charge states and for every single isotope in the mass spectrum. The algorithm selects the optimal set of isotopic distributions that best explain the mass spectrum. Monoisotopic masses are consequently identified with high confidence, enabling the de novo and sequence confirmation workflows to be applied with high efficiency. OmniWaveTM 0.25 0.25 0.25 20 20 20 0.25 0.25 0.25 20 20 20 0 10000 20000 30000 40000 50000 60000 70000 80000 0 1000 3000 4000 5000 6000 7000 8000 protein groups peptides ng K562 digest ng K562 digest 4257 4271 4483 7034 6913 7062 73465 73019 73472 28196 29019 29095 timsOmni: Excellent Sensitivity for Bottom-Up Proteomics Built on the robust foundations of the timsTOF Ultra 2, the timsOmni system seamlessly transitions to pass-through mode, delivering exceptional performance for bottom-up proteomics. This versatility allows for the analysis of single cells or other low input samples, down to a single immune cell, with unparalleled precision. Peptides and protein groups in a Promega K562 digest measured by dia-PASEF. A 22 min gradient was used with an Aurora Ultimate column at 250 nL/min fl ow rate. Results were searched against the Swissprot human database using Spectronaut 19 and directDIA+, with no matching applied between runs. Experience the best of both worlds: • Ultra-high sensitivity for bottom-up and top-down proteomics. • Switch effortlessly between advanced MSn eXd fragmentation modes and standard PASEF® methods. • Harness the diverse ion activation network for deep proteoform sequencing and advanced structural elucidation of a wide-array of biomolecules. It's a fact! The timsOmni provides access to all PASEF modes available on the timsTOF platform series. Online information www.bruker.com/ timsomni Bruker Switzerland AG Fällanden · Switzerland Phone +41 44 825 91 11 Bruker Scientifi c LLC Billerica, MA · USA Phone +1 (978) 663-3660 ms.sales.bdal@bruker.com - www.bruker.com Bruker Daltonics is continually improving its products and reserves the right to change specifi cations without notice. © BDAL 06-2025, 1920113, Rev. 01 Feature Ion Sources: • CaptiveSpray • NEOS (new off-line nanoESI source) • VIP-HESI (with built-in APCI) MSn eXd for multiple stages of ion activation and dissociation: Multi-stage trapped eXd fragmentation with charge state dependent reaction times and precise modulation of electron energy to access diverse fragmentation schemes. Ion enrichment technology for deep sequencing and structural elucidation: Signal amplification via ion enrichment and MSn eXd using omni-directional ion transfer for advanced ion processing workflows. Athena Ion Processor (AIP) for broader m/z range detection: Optimized release of ions depending on the application, addressing mass discrimination effects across a very wide m/z range and boosting sensitivity. Charge Data-Dependent Acquisition (cDDA): On-the-fly charge state deconvolution on LC time scales for intelligent quadrupole isolation of proteoforms across a wide dynamic range. Extended m/z range quadrupole isolation: <4500 m/z quadrupole isolation in transmission mode and 150 to >10,000 m/z omni-Q2 isolation in trapping mode The timsOmni - An eXtreme leap in deep proteoform sequencing and advanced structural elucidation For Research Use Only. Not for use in clinical diagnostic procedures.