Quantitative Proteomics Services To Upgrade Your Research

FACTSHEET 4D-DIA Quantitative Proteomics and 4D Phosphoproteomics Next Generation Ion Mobility Mass Spectrometry-based Proteomics Services 2 Next Generation Ion Mobility Mass Spectrometry-based Proteomics Services Overview Crown Bioscience, in partnership with leading third-party proteomics providers, offers next generation ion mobility-mass spectrometry (IM-MS)-based-proteomics services to global clients. This will allow clients to assess the biological function of proteins for understanding signalling mechanisms within cells as well as specific biomarkers to diseases, and systematically assess quantitative differences in protein profiles of distinct samples for biomedical and clinical research. Services Offered • 4D-DIA quantitative proteomics • 4D phosphoproteomics • Bundle package (4D-DIA Proteomics + 4D Phosphoproteomics) Applications • Global proteomics profiling of cells or tissues, with/without treatment • Proteomics biomarker discovery and validation • Drug mechanism of action and toxicity studies • Disease mechanism studies • Target identification and validation • Complementary analysis and correlation approaches for other omics analysis Our Advantages • Better reproducibility: no missing values, reduced batch effect, better data parallelism, and traceability • Increased sensitivity, wider dynamic range, and improved detection depth: identify more proteins from same or lower amount of sample, significantly improved quantification especially for low abundant proteins • Lower amount of samples required • Suitable for large sample cohort studies • Customized bioinformatic analysis available for large cohort studies Factors to Consider When Choosing Mass Spectrometry-based Proteomics Services Liquid chromatography (LC) coupled with MS has become the gold standard in various omics fields. When choosing proteomics services, there are several factors to consider: • Data completeness and reproducibility • Sensitivity and identification depth: • Number of identified and quantified proteins • Low abundant protein identification and quantification • Sample amount requirement • Speed, throughput, robustness and cost • Data analysis 4D-DIA Quantitative Proteomics: A Combination of 4D Proteomics and DIA Technology A new generation of 4D-DIA proteomics technology (Figure 1) combines 4D proteomics, which added ion mobility as the fourth separation dimension to traditional LC-MS/MS (retention time, mass-to-charge ratio (m/z) and MS/MS fingerprint), with data independent acquisition (DIA) strategy, which avoids data imbalance caused by randomness by realizing "lossless acquisition" of all possible data. + Higher sensitivity Deeper protein identification Better data reproducibility More reliable quantification Smaller sample size required 4D proteomics DIA 4D-DIA proteomics Figure 1: 4D-DIA proteomics 3 DDA-MS MS1 MS2 Survey scan & precursor selection m/z m/z Intensity Intensity Intensity DIA-MS Survey scan & across all isolation windows Fragmentation of selected precursors Fragmentation of all precursors in each window m/z Intensity m/z Cutting-edge 4D Proteomics Technology Classic LC-MS/MS-based bottom-up proteomics separate peptides based on 3 dimensions: chromatographic retention time (RT); m/z and ion intensity. Ion mobility analysis is a gas-phase technique allowing the separation of ions based on their mobility through an inert gas (typically helium or nitrogen) under the influence of an electric field. Adding ion mobility as an additional dimension of separation for peptide ions to LC-MS/MS analysis has become increasingly popular for proteomics studies (Figure 2). It significantly improves the scanning speed and detection sensitivity, as well as enhances proteomic analysis performance in terms of identification depth, detection cycle, and quantitative accuracy. 3 2 1 4 300 400 500 600 700 800 900 1000 1100 1200 m/z 0.7 BPC 400.0000-1000.0000 + Al MS FullScan 0.8 1 1.1 1.2 1.3 1.4 1.5 0.9 Intens. x10⁶ 2.0 1.5 1.0 0.5 0.0 0 10 20 30 40 50 60 70 80 90 100 Time [min] 4D Proteomics 1 — Retention time 2 — Mass-to-charge ratio (m/z) 3 — Intensity 4 — Ion mobility Data Dependent / Independent Acquisition (DDA / DIA) There are two data acquisition strategies in tandem mass spectrometry (MS/MS) data acquisition (Figure 3): DDA mode: The mass spectrometer selects a fixed number of most intense precursor ions. They are then fragmented and analyzed, in the second stage of tandem mass spectrometry. DIA mode: The mass spectrometer divides the full scan range of mass spectrometry into several windows and selects, fragments, and collects all ions in each window. DIA strategy avoids data imbalance caused by randomness by realizing "lossless acquisition" of all possible data. However, the "all-acquisition" strategy produces a highly complex spectrogram that poses a great challenge to data analysis. Figure 2: 4D proteomics Figure 3: DDA and DIA data acquisition 4 Technical Strengths of 4D-DIA Quantitative Proteomics 4D Alignment for Better Identification The greatest challenge in traditional DIA without ion mobility separation lies in the difficulty of reliably analyzing mixed spectra. During quantification, matching is mainly based on chromatographic elution time (retention time). However, due to co-elution, the retention time alone is not enough, and many other interfering signals will affect the detection accuracy. 4D technology provides ion-mobility separation and adds an extra dimension for calibration. It can accurately discriminate the specific peptide signals from the mixed spectra of DIA, effectively reducing spectra complexity and improving the detection accuracy and reliability of DIA (Figure 4). Nearly 100% Ion Utilization, Maximized Detection Sensitivity In the 4D-diaPASEF scanning mode, the ion-mobility-related CCS value correlates well with m/z. This feature allows the Quadrupole to scan gradually to collect nearly 100% of the ion signals, greatly enhancing the sensitivity and depth of detection (Figure 5). In comparison, in the traditional LC-MS/MS-based proteomics without ion mobility separation, only limited signals can be collected. Significant Improvement in Detection Depth 4D-DIA identifies and quantifies low-abundant proteins more accurately than traditional methods, thereby increasing the depth of proteomics detection (Figure 6). Better quantitative integrity 4D-DIA technology can further push the limits of sensitivity with advanced instrument performance and upgraded acquisition methods. Even at this trace level, 4D-DIA showed 85% data integrity for protein quantification (Figure 7). Figure 4: The chromatograms of EVGSHFDDFVTNLIEK peptide after calibration in traditional DIA (top) and 4D-DIA (bottom). There is a high interference signal in the traditional DIA method (top), while the signal background in the 4D-DIA (bottom) is much cleaner. Figure 6: >7,500 proteins can be identified from a single injection of 200 ng of HeLa lysate (120-minute run time), and 6,974 proteins can be quantified with 96% data completeness in triplicate runs. In comparison, conventional proteomics usually requires μg-level samples to detect about 5,000 proteins. Figure 7: >3,000 protein can be identified from a single injection of 10 ng HeLa lysate (120-minute run time), and 3,323 proteins can be identified by triplicated runs. While under the same study conditions, the 4D-DDA mode could only identify 2,723 proteins. Replicate Replicate 200 ng HeLa lysate 10 ng HeLa lysate Figure 5: 4D-DIA based on the 4D platform can reach nearly 100% ion utilization. Left–Data acquisition in 4D-DIA mode; Right–Comparison of ion utilization among 4D-DIA, DDA, and DIA. (Source: Florian Meier, et al. Nature Methods.) 4D-DIA Technology Effectively Reduces the Complexity of the Spectrum 600 2000 y11-1340.6733* y10-1193.6048* 500 1500 1000 500 1600 400 300 Intensity (10*3) Intensity 200 100 0 68.8 71.6 71.7 71.8 71.9 72.0 72.1 72.2 69.0 69.2 Retention Time Retention Time 69.4 69.6 69.8 y3-389.2395* y12-739.3697** 69.3 +1 ppm 12 9 b9-1034.4214* y7-816.4825+ y6-717.4141+ y4-502.3235+ b8-887.3530+ b10-1133.4896* b9-1034.4214+ 71.8 -2.6 ppm 69.8 -10.1 ppm 2000 340.6733* 93.6048* 1500 1000 500 1600 Intensity 71.6 71.7 71.8 71.9 72.0 72.1 72.2 Retention Time 69.8 9.2395* 39.3697** 9 34.4214* y7-816.4825+ y6-717.4141+ y4-502.3235+ b8-887.3530+ 133.4896* b9-1034.4214+ 71.8 -2.6 ppm 69.8 -10.1 ppm MS1 Fragmentation m/z MS2 m/z Ion Mobility 1/K0 diaPASEF scan 1.5 DDA 1.0 Aligned Retention Time (min) Abundance x 10⁴ (a.u.) 0.5 16.5 0.0 16.6 16.7 DIA diaPASEF 1 2 3 Total 8,000 7,000 Proteins 6,000 5,000 4,000 3,000 2,000 1,000 0 1 2 3 Total 3,500 3,000 Proteins2,500 2,000 1,500 1,000 500 0 5 Isomerization Brings Site Identification Challenge for Phosphoproteomics Phosphorylation is a common type of post translational modifications (PTM), with more than 30% of proteins in cells phosphorylated. Phosphorylation is the process of transferring phosphate groups from ATP or GTP to specific sites (usually Ser, Tyr, Thr) of proteins, catalyzed by phosphorylating kinase (Figure 8). It is one of the most fundamental, prevalent, and important mechanisms to regulate and control protein activity and function, and is involved in various physiological and pathological processes, regulating cellular activities such as proliferation, development, differentiation, and apoptosis. Phosphorylation on different sites on the same peptide can lead to phosphopeptide isomers co-elution on chromatography (Figure 9). These co-eluted phosphopeptide isomers cannot be separated on conventional LC-MS/MS platforms. According to available statistics, at least 26% of phosphopeptides have isomers, 50% of which cannot be effectively differentiated on chromatography, making it difficult to identify phosphorylation sites. Signal input Signal output Enzyme State ‘B’ PTM crosstalk Localization Activity Conformation Binding Turnover State ‘A’ Enzyme P P PP ATP P ADP P P P P Kinase Me P Ub Ac P P Histogram Phosphopeptide Positional Isomer RT Difference B Co-elute fragment B Co-elute ferential gmentation B Co-elute onclusive gmentation Bferential RT 60% 50% 8% 6% 6% 4% 3% 3% 3% 3% 2% 2% 1% 1% 1% 1% 1% 1% 1% 1% 2% 50% Retention Time Difference Between Positional Isomers (min) Percentage of Isomer Pairs 40% 30% 20% 10% 0% 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25 4.5 4.75 5 5.25 5.5 5.75 6 6.25 6.5 6.75 7 7.25 7.5 7.75 8 >8 Phosphopeptide Positional Isomers Figure 8: Phosphorylation Figure 9: Isomerization brings site identification challenge for phosphoproteomics 6 4D Phosphoproteomics Features • Immobilized metal affinity chromatography (IMAC) strategy: using proprietary targeted antibodies to enrich phosphopeptides, to reduce sample complexity • Additional ion mobility separation enables more reliable and deeper coverage for phosphorylation • Strict dual quality control to remove low confident data: false discovery rate (FDR, 1%); false localization rate (FLR, 0.75) • Upgraded bioinformatic analysis available: kinase prediction, signaling analysis, and data mining Technical Strengths of 4D Phosphoproteomics Deeper Coverage of Phosphorylation Site Identification 4D technology brings a dramatic increase in detection depth and sensitivity. The timsTOF Pro system can acquire up to 100 MS/MS spectra per 1.1 second scanning cycle. >10,000 phosphorylation sites (localization probability > 0.75) were identified with high confidence in various cell tissues, 50% higher than the traditional method (Figure 10). More Reliable Modification Identification Results 4D phosphoproteomics resolves the issue of isomerization in PTM through ion mobility separation, which ensures more reliable identification of PTMs. As shown in Figure 11, the elution time and m/z ratio of many phosphorylated peptide isomers are identical, but the structural differences of the phosphorylation locations can be effectively detected by ion mobility. The separation of both ion mobility and HPLC can maximize the identification and differentiation of these co-eluted modified peptide signals. Protein extraction and enzymolysis Enrichment of phosphorylated peptides Pre-separation LC-MS/MS Site identification and quantification Phosphoproteomic data mining 4D Phosphoproteomics Quantitative Analysis Workflow Figure 10: Deep coverage of phosphorylation Figure 11: Adding ion mobility as the 4th separation dimension help to identify co-eluted modified peptides site identification 0.0 0.2 0.4 Localization Probability Confidence 0.6 0.8 1.0 25 0.75 FLR threshold =0.75 Low High # Phosphorylation Sites (x1000) 20 15 10 5 0 Human Stomach Tissue Mouse Brain Tissue HeLa 293T 18000 16000 Phosphosites 14000 12000 10000 8000 6000 4000 2000 0 0.0 0.2 0.4 Localization Probability Confidence 0.6 0.8 1.0 25 0.75 FLR threshold =0.75 Low High # Phosphorylation Sites (x1000) 20 15 10 5 0 Mouse Brain issue HeLa 293T Mass Spectrometry Core Facility • Top mass spectrometers such as Bruker timsTOF Pro/ timsTOF Pro 2 series, and Thermo Fisher Scientific Orbitrap Exploris™ 480 • Additional separation dimension by ion mobility separation to traditional LC-MS/MS based proteomics • Bruker Trapped Ion Mobility Spectrometry (TIMS) • Thermo Fisher Scientific FAIMS Sales US: +1 858 622 2900 UK: +44 870 166 6234 Get in touch Science busdev@crownbio.com consultation@crownbio.com www.crownbio.com • Unique diaPASEF technology to dramatically increase data acquisition speed • Evosep One provides robust and excellent chromatographic separation • Continuous analysis of thousands of samples to provide stable performance without cleaning, enhancing the stability of batch analysis