AssayMAP Automated Protein Sample Prep for Mass Spectrometry Rachel Bolger, Product Manager [email protected] 4/11/2014 Confidentiality Label May 5, 2014 1 The image part with relationship ID rId17 was not found in the file. The LC/MS Influence For Research Use Only. Not for use in diagnostic procedures. May 5, 2014 2 The image part with relationship ID rId17 was not found in the file. The Vision A complete menu of automated methods for protein analysis • • • with method-specific cartridges on a single instrument with simplified user-interface Heavy Chain Pyroglutamate Disulfide shuffling Light Chain Fab Deamidation/oxidation Glycosylation site Fc Intact molecular weight Peptide Mapping Glycan Analysis For Research Use Only. Not for use in diagnostic procedures. May 5, 2014 3 The image part with relationship ID rId17 was not found in the file. AssayMAP Technology Components Precision & Increased Throughput AssayMAP Bravo Liquid Handling platform provides precision pipetting and parallel processing Robust & Reproducible quantitative chromatographic separation method provides highly reproducible results Easy to Use Standardized user interfaces enable control over key assay parameters, are easy to use and make methods simple to transfer For Research Use Only. Not for use in diagnostic procedures. May 5, 2014 4 The image part with relationship ID rId17 was not found in the file. AssayMAP Technology - Components The image part with relationship ID rId17 was not found in the file. Why Chromatography? AssayMAP is like microscale HPLC columns, run 96 at a time, yielding chromatographic-like performance Traditional Pipet Tip Column AssayMAP Cartridge VS Shower Bath • Multi-pass contact (equilibrium adsorption) • Single-pass contact (chromatography!) • No flow rate control • Precise, controlled flow rate • Non-quantitative extraction • Quantitative binding & elution • Less efficient removal of contaminants • Efficient removal of contaminants • Recover < 100 % of capture • Recover 100% of capture • Diluted >10 X • Minimal volume (10 µL elution) For Research Use Only. Not for use in diagnostic procedures. May 5, 2014 6 The image part with relationship ID rId17 was not found in the file. AssayMAP Technology: Cartridge Portfolio Applications (Apps) Ab Capture Protein Digestion Peptide Clean Up Fractionation Protein A and G Cartridges (protocol only - no cartridge) C18 & RP-S Cartridge SCX, C18 & RP-S Cartridge Coming soon Workflows: combine individual Apps into powerful workflows Protein Sample Prep for mass spec analysis Purify Digest Phosphopeptide Enrichment with TiO2 Cleanup Immunoaffinity Purification N-glycan Sample Prep for HPLC, CE or mass spec analysis Release and Label Glycans The image part with relationship ID rId17 was not found in the file. Software Designed for Scientists Flexible and intuitive software, out of the box Workflow-specific control parameters Standardized deck layout “Pushbutton” run controls For Research Use Only. Not for use in diagnostic procedures. May 5, 2014 8 The image part with relationship ID rId17 was not found in the file. Software - Calculator Each input in calculators is described in full user guides The image part with relationship ID rId17 was not found in the file. Protein Purification App • Protein A, Protein G - Quantitative single pass binding, washing and elution - Sample volumes from 10-1000 µL - Elution volumes as low as 25 µL - Ability to wash with multiple stringency wash solutions The image part with relationship ID rId17 was not found in the file. Protein Purification App - User Interface For Protein A (PA-W), Protein G (PG-W), Streptavidin (SA-W) The image part with relationship ID rId17 was not found in the file. Protein Purification App Quantitative Recovery Quantitative recovery >95% Less than 2% CVs 30 min/96 samples Minimal hands on time Very reproducible, CVs = <5% Integrates with other workflows Elution in < 25µl For Research Use Only. Not for use in diagnostic procedures. May 5, 2014 12 The image part with relationship ID rId17 was not found in the file. Peptide Sample Prep Workflow In-Solution Protein Digestion Peptide Cleanup Peptide Fractionation The image part with relationship ID rId17 was not found in the file. In-Solution Digestion App 1-3 hrs 2-18 hrs Input: Output: • • • • • Particulate-free solutions of proteins Up to 384 samples -(four) 96-well plates Sample plate from a Purification protocol Sample volume dependent on chemistry conditions – Use the Reagent Calculator! • Sample plates contain digested proteins = peptides Samples now ready for peptide cleanup Dramatically reduces hands on time and human variability The image part with relationship ID rId17 was not found in the file. In-Solution Digestion App – User Interface The image part with relationship ID rId17 was not found in the file. Peptide Cleanup App C18 or RP-S Input: Output: • • • • • Particulate-free, aqueous solutions of peptides Sample plates from In-Solution Digestion protocol Up to 96 samples (1 plate) Up to 250 µL sample load Up to 25x concentration factor!!! • Salt-free, solutions of peptides in organic solvent Elution volume as little as 10 µL The image part with relationship ID rId17 was not found in the file. Peptide Cleanup App – User Interface The image part with relationship ID rId17 was not found in the file. Reversed-phase cartridge characterization BSA load capacity study RH = relative hydrophobicity The image part with relationship ID rId17 was not found in the file. BSA digestion and cleanup Person to person reproducibility Low elution volume = powerful enrichment Low intra- and interday CVs For Research Use Only. Not for use in diagnostic procedures. May 5, 2014 19 The image part with relationship ID rId17 was not found in the file. Peptide Fractionation SCX, C18, or RP-S Input: Output: • • • • • Particulate-free, solutions of peptides Sample plates from Peptide Cleanup protocol Up to 96 samples (1 plate) Up to 250 µL sample load • • Solutions of peptides in various solvents/salts As many as 6 fractions can be collected Can collect small volume fractions (~ 5 µL) The image part with relationship ID rId17 was not found in the file. Fractionation - User interface The image part with relationship ID rId17 was not found in the file. Fractionation AssayMAP Bravo 1290 Infinity LC with AdvanceBio Peptide Mapping Column 6550 iFunnel Q-TOF with Dual Agilent Jet Stream ESI Source Confidentiality Label May 5, 2014 22 The image part with relationship ID rId17 was not found in the file. Fractionation – Optimized peptides elute mostly in one fraction Confidentiality Label May 5, 2014 23 The image part with relationship ID rId17 was not found in the file. Fractionation – Needs More Optimization peptides elute across multiple fractions Confidentiality Label May 5, 2014 24 The image part with relationship ID rId17 was not found in the file. E. coli fractionation by SCX Elution by increasing ionic strength (KCl) The image part with relationship ID rId17 was not found in the file. Fully automated AssayMAP Bravo Workstation for peptide sample prep BenchBot Robot AssayMAP Bravo Plate Hub XPeel Multidrop Combi INHECO Incubator MP Centrifuge PlateLoc The image part with relationship ID rId17 was not found in the file. AssayMAP Automated Protein Sample Prep Platform Powerful enrichment due to small volume Total workflow reproducibility Dramatically reduces human variability Standardized interfaces are easy to use Out-of-the box automation Integrated workflows For Research Use Only. Not for use in diagnostic procedures. May 5, 2014 27