SpectroDive NEXT GENERATION TARGETED PROTEOMICS. Integration of Ready-Made Panels Improved Workflow for Custom Panels

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1 SpectroDive NEXT GENERATION TARGETED PROTEOMICS Integration of Ready-Made Panels Improved Workflow for Custom Panels

2 IMPROVING MULTIPLEXING FOR TARGETED PROTEOMICS Novel targeted proteomics workflows can be highly multiplexed, allowing for parallel quantification of hundreds of proteins. This requires new approaches for the development and handling of optimized assay panels. Targeted proteomics is the best analytical approach when a selected set of proteins needs to be quantified with high sensitivity, quantitative accuracy, and reproducibility. Previously, most applications used only small assay panels, limited to tens of proteins. The main reasons for under-using the multiplexing capabilities of modern PRM and MRM are manual assay generation methods that are suitable for single proteins, but not hundreds. Biognosys offers two solutions to turn targeted proteomics into a highly multiplexed technology: Ready-made assay panels that don t need any development, and a simplified workflow for generation of large custom panels; both supported by the latest version of SpectroDive. With routine investigations of plasma samples in mind, Biognosys developed PQ5: a ready-made kit including stable isotope standards for the targeted analysis of more than 5 proteins for human plasma profiling. Integration of PQ5 into SpectroDive provides researchers with a largely expanded panel of proteins. Supported Instruments With the latest version of SpectroDive, Biognosys has introduced a workflow for quick set-up of optimized custom-made panels. With the panel set-up wizard, users can quickly assemble an optimized assay panel from the peptides that are most suited for quantification. Additional features of SpectroDive include: MS1 support for PRM: Improved confidence in peak picking by using MS1 information MaxQuant.Live support: Analysis of MaxQuant.Live acquired data; inclusion list export in MaxQuant.Live compatible format False discovery rate control: Increased confidence in peptide identification, specifically in larger panels Pulsar integration: Fast and scalable library generation with Biognosys embedded search engine Easier assay panel generation: Intuitive and automated selection of the best suited peptides for any target protein assay Minimum System Requirements FEATURES Full automation High throughput Optimized scheduling Powered by Pulsar Custom-made panels PQ5 support BENEFITS «We use SpectroDive 9. to perform highly multiplexed targeted proteomics experiments. The High-Precision irt concept expedites our newly introduced global targeting approach based on MaxQuant.Live, which enables targeted proteomics with thousands of peptides.» Florian Meier, Max Planck Institute of Biochemistry, Martinsried, Germany (Matthias Mann Group) Publication-ready results with minimum effort More samples, more meaningful results without the need of manual revision Increase your number of targets with High- Precision irt Calibration assisted scheduling Fast and scalable library generation for a library-based panel generation Easy and intuitive panel generation wizard Native support for our ready-made panel for the absolute quantification of more than 5 human plasma proteins Thermo Scientific Q Exactive Series Thermo Scientific Orbitrap Fusion Series Thermo Scientific TSQ Series SCIEX TripleTOF Series SCIEX Triple Quad Series SCIEX QTRAP Series SCIEX API Series Agilent 64 Series Waters Xevo TQ-S Windows 7 x64 Intel Core CPU 2.7 GHz (quad core) or similar 5 GB of free HDD space 16 GB of RAM memory.net Framework 4.7 Better user experience Seamless pipeline Setup wizards, comprehensive plots and reports, list of candidates ready to export From panel generation to biological interpretation in one application

3 RELIABLE PEAK PICKING WITH CUSTOM PANELS Targeted proteomics is known to be the most flexible tool for the quantification of multiple candidate proteins in biological and biomedical Color Key applications. Often these assay panels are custom-made to address unique research questions. SpectroDive now supports the optimization of custom panels and subsequent data analysis. PQ5: A READY-MADE PANEL FOR HUMAN PLASMA Targeted proteomics has been limited by the complexity of developing large customized panels. PQ5 is Biognosys ready-made panel that allows for the identification and quantification of more than 5 proteins commonly used for human plasma profiling. SpectroDive is optimized to utilize PQ5 as a large ready-made panel for liquid biopsies. Original Heatmap Cell Cycle Value PHD1 MSN4 GAT1 SWI4 YOX1 CLB5 PCL1 CLN2 TOS4 HCM1 PLM2 NRM1 CIN8 PDS1 NDD1 FHL1 FKH2 MSN2 SWI6 MBP1 IXR1 SWI5 ACE2 SFG1 ASH1 MCM1 SIC1 Recently, Kelliher et al. (218) 1 investigated the role of transcription factors in the periodic expression of genes and the subsequent production of proteins during the cell cycle. In their study, Kelliher et al. used budding yeast Saccharomyces cerevisiae as a model system, and opted for targeted quantitative proteomics to understand transient protein expression patterns over the course of the cell-cycle. The researchers developed custommade assays for 45 cell cycle proteins and were able to detect 31 unique proteins dynamically expressed during the cell cycle. Heatmap with SpectroDive Cell Cycle WTR1.R1 WTR1.R2 WTR1.R3 WTR1.R4 WTR1.R5 WTR1.R6 WTR1.R7 WTR1.R8 WTR1.R9 WTR1.R1 WTR1.R11 WTR1.R12 WTR1.R13 WTR1.R14 WTR1.R15 WTR1.R16 WTR1.R17 WTR1.R18 WTR1.R19 WTR1.R2 Z-Score: To test SpectroDive s ability to quantify the peptides of interest, Biognosys re-analyzed the published dataset. Using SpectroDive's panel generation functionality and under default factory settings for analysis, we were able to reproduce the identical protein expression pattern over the cell cycle, reaching the same biological interpretation with a minimum amount of time and resources. 1) Christina M. Kelliher, et al. Layers of regulation of cell-cycle gene expression in the budding yeast Saccharomyces cerevisiae. Molecular Biology of the Cell. 218, 29, PHD1 MSN4 GAT1 SWI4 YOX1 CLB5 PCL1 CLN2 TOS4 HCM1 PLM2 NRM1 CIN8 PDS1 NDD1 FHL1 FKH2 MSN2 SWI6 MBP1 IXR1 SWI5 ACE2 SFG1 ASH1 MCM1 SIC1 qvalue (-Log1) Gbyte minutes Cancer Plasma Study Fold Change Cancer / Healthy Peak Memory Usage PRM run Analysis Time PRM run Subset PQ5 (16 peptides) Full PQ5 (1'6 peptides) PRM runs 225. CRP S1A9 S1A PRM runs SAA1 Human plasma is a widely used matrix in clinical and biological studies. It allows the researcher to gain comprehensive insights into a body s health status and can easily be collected from patients and healthy individuals. PQ5 contains more than 8 carefully selected stable-isotope standard (SIS) labeled peptides, corresponding to more than 5 proteins commonly found in human blood plasma. Efficient scheduling workflows in SpectroDive 9., together with the optimized PQ5 assay panel, allows the targeted interrogation of patient samples at the level of broadness of a discovery workflow. The figure on the left (top) shows differentially abundant proteins in human plasma detected with the PQ5 panel, including inflammation markers known to be elevated in cancer; e.g. C-reactive protein and protein S1-A9. SpectroDive includes quality control parameters commonly used in discovery proteomics that increase the researcher s confidence of identifying the correct peaks, such as FDR control and MS1 support for PRM. Its algorithms are computationally optimized to handle even the largest projects without limitations in assay panel size. The figures on the left demonstrate SpectroDive 9. is highly scalable towards experiment size, irrespective of how many peptides are targeted in the panel. In the examples shown, two-hour PRM runs acquired on a high-resolution instrument have been analyzed.

4 FROM PANEL REFINEMENT TO BIOLOGICAL INTERPRETATION SpectroDive takes the hassle from targeted proteomics by offering integrated panel optimization for custom panels, support for readymade panels, High-Precision irt Calibration-assisted scheduling and ready-to-publish results without manual revision. SpectroDive's custom panel generation tool is more intuitive and effortless than ever. Generate your panel using a library as a base: follow SpectroDive's wizard to obtain your best fitting panel. SpectroDive, powered by Pulsar, provides a seamless workflow: use your run files as input to generate a library. Optimize your workflow with library assisted panel generation. Pulsar can search DDA, DIA and PRM (with MS1 information) data. SpectroDive's peak picking is very robust and rarely requires manual adjustments. In the Analysis Perspective, informative overview plots ranging from the individual sample to the entire data set can be used to confirm correctness of peak picking. High-Precision irt Calibration allows a refined scheduling for increased multiplexing. Readymade assay panels such as PQ5 benefit the most from this feature. Use your panel to analyze your runs with the default factory settings. Get your list of candidates in the Post Analysis Perspective with no further manual revision.

5 HOW TO GET SPECTRODIVE If you are interested in learning more about SpectroDive or want to get your free trial license, don t hesitate to contact us at order@biognosys.com. We would be pleased to schedule a meeting with you and support you in implementing next generation proteomics into your laboratory. Biognosys is a leader in next generation proteomics, dedicated to transforming the life sciences by making the most advanced proteomics tools available to researchers. Biognosys offers a suite of products and services to decode the proteome and equip researchers from all fields with a deep view of protein expression and quantification in a biological system.