Bravo with AssayMAP. Automated Sample Preparation

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1 Bravo with AssayMAP Automated Sample Preparation

2 Why Automate? At some point, you cannot do it well without automation! Maintain data quality with increased sample capacity. Consistency scales better with automation.

3 Bravo Automated Liquid Handling Platform Innovative Engineering Space-saving design Fast, quiet operation Unique size-sensing plate gripper enables flexible automation Rapid-swap heads offer ultimate flexibility and future upgradeability Open strut-free design permits use in laminar flow hoods and high integration accessibility Best in class liquid handling speed, precision, and accuracy Clean base with no below-deck Electronics permits easy accessory integration and deck customization

4 Multiple Formats With One Head 96, 384 and 1536 Well Format Pick up Single or Multiple Columns and Rows from a full tip box Pick up Single tip to Perform Hit Picking

5 AssayMAP Bravo Technology Components For Mass Spec Analysis Target Customer Simplified User-interface Digestion Cleanup Fractionation AssayMAP Cartridges: Cleanup: C18 RP-S Fractionate: RP-S SCX Bravo Liquid Handling Platform

6 Software

7 AssayMAP Peptide Sample Prep Workflow Feature Set Feature Set AssayMAP In-Solution Digestion Automated protocol with minimal manual steps, that processes up to four 96 well plates with user-supplied reagents. Labware is provided in starter kits AssayMAP Peptide Cleanup Cleanup one 96 well plate of peptide samples using reversed phase cartridges. Elute in 10 μl volume, allowing minimal to no dry down times. Uses AssayMAP C18 or RP-S cartridges AssayMAP Fractionation Simplify one 96 well plate of complex samples, offline from LCMS, using stepwise ph or salt elution to generate up to 6 fractions. Uses AssayMAP C18 or RP-S cartridges Benefits: Dramatically improves Mass Spec throughput Automation brings high reproducibility/ low %CVs more consistent & reliable data Reduce solvent use for LC minimum of 5x Fractionation- simplifies & accelerates method development from 2 weeks to 1 day by testing 96 methods simultaneously

8 mau mau Automated Tryptic Digestion BSA 200 Overnight In-Solution Undigested BSA 200 Denaturation Reduction Alkylation Proteolytic Digestion min TR-W Cartridge Minutes independent runs (2 cartridge lots) 5 µl/min 4.8 mg/ml BSA 480 µg load

9 AssayMAP Peptide Cleanup Advance BioPeptide Mapping column AssayMAP Sample Prep Infinity + LC 6550 ifunnel Q-TOF AssayMAP Bravo was used to denaturate, reduce, alkylate, and digest BSA in Urea (n=64, 62) and Guanidine HCl (n=64, 64) in parallel on two different days Samples were acidified and cleaned using AssayMAP C18 cartridges (day 1) and AssayMAP RP-S cartridges (day 2). Samples were lyophilized, resuspended, and analyzed using an Agilent AdvanceBio Peptide Mapping column on an Agilent 1290 Infinity LC and then analyzed using an Agilent 6550 ifunnel Q-TOF mass spectrometer.

10 AssayMAP Data Total workflow reproducibility Total workflow CVs < 4% Agilent AssayMAP Peptide Sample Prep Agilent AdvanceBio Peptide Mapping column Agilent 1290 Infinity LC Agilent 6550 ifunnel Q-TOF mass spectrometer The AssayMAP workflow took 2 hours hands time each day Scatter plot showing the peak area of 4 peptides across 128 data points (64 replicates; repeated over 2 days)

11 AssayMAP Data Target enrichment using fractionation Simplify Sample into discrete fractions (up to 6) Used KCl to serially fractionate protein mixture using AssayMAP SCX column. The data shows that 67.1% of the peptides segregated uniquely enabling better identification of proteins in a complex mixture

12 AssayMAP Sample Prep Solution HPLC-like performance on a liquid handling platform Feature Set Total workflow reproducibility Parallel processing for rapid method development Dramatically reduces human variability, hands on time Increases Mass Spec throughput Standardized interfaces are easy to use Out-of-the box solution no automation engineer Integrated workflows, simple method transfer