AssayMAP Peptide Cleanup Protocol Guide

Transcription

1 AssayMAP Peptide Cleanup Protocol Guide Original Instructions

2 Notices Agilent Technologies, Inc No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or translation into a foreign language) without prior agreement and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws. Manual Part Number G Edition Revision 01, July 2013 Contact Information Agilent Technologies Inc. Automation Solutions 5301 Stevens Creek Blvd. Santa Clara, CA USA Technical Support: (US only) or (rest of the world) service.automation@agilent.com Customer Service: or orders.automation@agilent.com European Service: +44 (0) euroservice.automation@agilent.com Documentation feedback: documentation.automation@agilent.com Web: automation Acknowledgements Microsoft and Windows are either registered trademarks or trademarks of the Microsoft Corporation in the United States and other countries. Warranty The material contained in this document is provided as is, and is subject to being changed, without notice, in future editions. Further, to the maximum extent permitted by applicable law, Agilent disclaims all warranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchantability and fitness for a particular purpose. Agilent shall not be liable for errors or for incidental or consequential damages in connection with the furnishing, use, or performance of this document or of any information contained herein. Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms, the warranty terms in the separate agreement shall control. Technology Licenses The hardware and/or software described in this document are furnished under a license and may be used or copied only in accordance with the terms of such license. Restricted Rights Legend If software is for use in the performance of a U.S. Government prime contract or subcontract, Software is delivered and licensed as Commercial computer software as defined in DFAR (June 1995), or as a commercial item as defined in FAR 2.101(a) or as Restricted computer software as defined in FAR (June 1987) or any equivalent agency regulation or contract clause. Use, duplication or disclosure of Software is subject to Agilent Technologies standard commercial license terms, and non- DOD Departments and Agencies of the U.S. Government will receive no greater than Restricted Rights as defined in FAR (c)(1-2) (June 1987). U.S. Government users will receive no greater than Limited Rights as defined in FAR (June1987) or DFAR (b)(2) (November 1995), as applicable in any technical data. Safety Notices WARNING A WARNING notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in personal injury or death. Do not proceed beyond a WARNING notice until the indicated conditions are fully understood and met. CAUTION A CAUTION notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in damage to the product or loss of important data. Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met. AssayMAP Peptide Cleanup Protocol Guide ii

3 Contents Preface... 4 About this guide... 4 Reporting problems... 4 Before you begin... 5 Equipment... 5 Kits and labware requirements... 7 Reagent requirements... 7 Cartridge use and storage guidelines... 8 Preparing your samples... 9 Sample particulates... 9 Sample composition... 9 Sample load volume... 9 Sample plate setup Preparing your reagents Using the Peptide Cleanup Calculator Preparing reagents for the Peptide Cleanup protocol Setting up a Protocol Starting up Installing software and opening the application interface Opening the protocol and app Priming the wash station and initializing the Bravo platform Running a protocol Assay Development guidelines Automation Protocol Steps AssayMAP Peptide Cleanup Protocol Guide 3

4 Preface About this guide This guide is designed for both routine operators and assay developers. This guide does not cover how to set up and maintain the Bravo Platform, detailed safety guidelines, details about the Bravo 96AM Head, details about the Pump Module, or detailed editing of the protocol or application interface in the VWorks software. For information on these topics, go to the Automation Solutions knowledge base at and click the link for PDF user guides. Reporting problems If you find a problem with the AssayMAP Peptide Cleanup solution, contact Automation Solutions Technical Support. For contact information, see Notices on page ii. For reporting problems with... Have the following information ready... Hardware Software Protocol Guide Device serial number from the Bravo serial number label Short description of the problem Relevant software version number (for example, automation control software, diagnostics software, and firmware) Error message Relevant files, such as log files Send your comments in an to documentation@agilent.com AssayMAP Peptide Cleanup Protocol Guide 4

5 Before you begin Introduction The AssayMAP Peptide Cleanup solution enables automated cleanup of 8 to 96 peptide samples in a single run. The solution consists of the following components: Agilent Bravo Platform for AssayMAP (liquid handler) Agilent AssayMAP microchromatography cartridges packed with either a 20 μm diameter silica resin with 150 Å pores derivitized with C18 (C18) or a 20 μm diameter underivitized polystyrene polystyrene hydrophobic reversed-phase resin with 100 Å pores (RP-S). Agilent software and supporting files (VWorks software, Launch Pad, AssayMAP Peptide Cleanup protocol, and application interface) Equipment Table AssayMAP Bravo Platform requirements Component Part number Bravo Platform for AssayMAP technology Includes the base Bravo Platform, computer, and the following: Gripper Upgrade Bravo 96AM Head 96AM Wash Station Pump Module AM Cartridge & Tip Seating Station Custom Plate Nest Risers, 146 mm Peltier Thermal Station with STC controller Orbital Shaking Station with Control Unit Red PCR Plate Insert Two 10-L carboys G5542A G5498B#046 G5498B#057 G5498B#058 G G5498B#017 G5498B#055 G5498B#035 G5498B#033 G5498B#013 NA AssayMAP Peptide Cleanup Protocol Guide 5

6 Figure AssayMAP Bravo Platform and accessories Table Optional Equipment Centrifuge with microplate rotor SpeedVac PlateLoc Software This guide documents the following versions, which are required for the Bravo 96AM Head: VWorks Automation Control Bravo Diagnostics Bravo Firmware AssayMAP Launch Pad v3.0 NOTE You can download the most recent version of the installer from AssayMAP Peptide Cleanup Protocol Guide 6

7 Kits and labware requirements RP-S and C18 reverse phase cartridge types are currently available for performing peptide cleanup on the AssayMAP Bravo. Each of these cartridge types can either be purchased as a rack of 96 cartridges plus the labware required for peptide cleanup and in solution digestion or as an individual rack of 96 cartridges. Table AssayMAP Digestion and Cleanup Starter kits AssayMAP Digestion and RP-S Cleanup Starter Kit AssayMAP Digestion and C18 Cleanup Starter Kit G reversed-phase (RP-S) cartridges plus labware for In- Solution Digestion and Peptide Cleanup G C18 cartridges plus labware for In-Solution Digestion and Peptide Cleanup AssayMAP cartridge racks* G AssayMAP C18 cartridge rack (Qty 96) AssayMAP RP-S cartridge rack (Qty 96) * Labware is included in Starter Kits, but required for In-Solution Digestion A and Peptide Cleanup B when using individual racks mL Deep-Well Plates (AbGene AB-1127) A Well Round Bottom, White Plates (Greiner ) A µl Pipette Tips (Agilent ) A 4 12-Column Low-Profile Reservoirs (Seahorse ) B 2 96-Well Round-Bottom, Clear Plates (Greiner ) B 3 96-Well PCR Plates (Eppendorf ) A,B CAUTION The supplied protocol and app support a specific list of labware only. Using undefined or incorrectly identified labware on the Bravo deck can cause a crash and potential damage to the Bravo 96AM Head. For more details contact technical support. Reagent requirements The simplest deck configuration for the Peptide Cleanup has three solvents on the Bravo deck: 50% ACN:50% H2O:0.1%TFA, 100% H2O:0.1%TFA, and deionized water Table Required solvents to be supplied by customer Solution Quality Estimated Volume* acetonitrile (ACN) 100%, HPLC-grade ~100 ml trifluoroacetic acid (TFA) 100%, HPLC-grade ~0.3 ml purified deionized water (H2O) HPLC-grade, MilliQ, or equivalent ~5 L * Use provided Reagent Calculator for more accurate volumes and suggested overages. AssayMAP Peptide Cleanup Protocol Guide 7

8 Cartridge use and storage guidelines Follow these guidelines to get the best performance from the AssayMAP cartridges: Use only particulate-free samples, from a previous chromatographic run or by filtration (0.22 μm). Use only primed and equilibrated cartridges. We guarantee performance when you use AssayMAP cartridges on the same day you equilibrate them. Do not allow wetted cartridges to dry out. We do not guarantee performance of stored cartridges following equilibration. Store unused cartridges in the original shipping package at room temperature. Cartridges stored in this manner are stable for 6 months from the date of purchase. AssayMAP Peptide Cleanup Protocol Guide 8

9 Preparing your samples Sample particulates Samples must be free of macromolecular particulates, such as large protein aggregates and cellular debris. You may load any sample previously passed through another type of chromatography column or a 0.22 μm filter onto an AssayMAP cartridge. Sample composition What are optimal ph conditions? One of the most important considerations for highly efficient and unbiased binding of peptides to the reversed-phase resin is the ph of the sample, which should be acidic (ph<3) for standard, low-ph peptide cleanup with C18 or RP-S cartridges or basic (> ph 10) with RP-S cartridges. Peptides are amphoteric molecules with a diverse range of physiochemical properties. For some classes of peptides, cleanup under high-ph conditions may help to promote retention (e.g., highly basic peptides) or preserve acid-labile modifications (e.g., histidine phosphorylation). You should acidify samples before loading onto AssayMAP peptide cleanup cartridges, generally by adding TFA, formic acid, or acetic acid. You can use the Single Liquid Addition utility to perform this addition. What sample components cause concerns? Samples containing organic solvents or some types of detergents should be avoided as they may bias the peptides that bind the column. For example, loading samples in a buffer containing greater than 5% acetonitrile will inhibit binding of hydrophilic peptides. Silica-based C18 cartridges are at risk with a sample ph higher than 8. If you have concerns about a specific buffer component, you should examine scientific literature for the known effects of this type of molecule on reversed-phase resins. You can use the Single Liquid Addition utility to modify the composition of your samples with a dilution or ph adjustment, especially after using the In-Solution Digestion workflow. Sample load volume AssayMAP cartridges can accommodate volumes ranging from 10 to 245 μl. What is the binding capacity of the cartridge? For each AssayMAP peptide cleanup cartridge, the quantitative binding capacity depends on the relative hydrophobicity of the peptides in the sample and the sample complexity. When examining human insulin in isolation, the quantitative binding capacity is approximately 400 µg per peptide cleanup cartridge. When examining a tryptic digestion of BSA, the quantitative binding capacity is approximately 150 (without loss of hydrophilic peptides) to approximately 300 µg (with some loss of highly hydrophilic peptides) for this complex mixture. The total binding capacity (supersaturating amounts) is the maximum mass of peptide that can bind to the cartridge and is greater that the quantitative binding capacity. Binding mass can be increased if quantitative binding is not required. What is the concentration of the target in the sample? Use this formula to determine the volume of sample to load: μl sample to load = μg peptide desired μg/μl of peptide in sample AssayMAP Peptide Cleanup Protocol Guide 9

10 Sample plate setup How many samples can I process? You can process 8 to 96 samples in parallel. The AssayMAP Probe Syringes always move in unison. The position of samples in their microplate dictates the positions needed for cartridges in the 96AM Cartridge & Tip Seating Station, and the reservoirs for solutions at the other deck locations. How do I need to configure fewer than 96 samples in a microplate? When you use only a subset of wells in a sample microplate, fill the same subset of positions at all the deck locations. The default settings for the AssayMAP platform assume that samples will be arranged in multiples of 8 in a column-based configuration. The AssayMAP Bravo applies differential pressure to seat cartridges based upon the number of full columns of cartridges. Therefore, to achieve proper cartridge seating entire columns must be used to guarantee optimum performance. The kits supply either an Eppendorf PCR plate or a Greiner 96-Well Round Bottom, clear plate for sample set up. A full list of acceptable labware is available in the dropdown menu of the application interface. Figure Sample plate layout and reservoir recommendations If the number of samples you have is not a multiple of 8, use resin free AssayMAP cartridges (Agilent part number G ) to fill the empty column/row positions. This will prevent liquids from dripping on the deck or being dispensed on the deck during the cup washing step. How much excess sample do I need to load in a microplate well? Pipette an excess of at least 10 µl per well of extra volume for PCR plates, 20 µl for U-bottom and Deep Well plates and at least 3 mls excess per channel of a 12 channel reservoir to ensure that the sample does not fully deplete, which would result in aspiration of air into the cartridges. Modify this volume accordingly if evaporation due to volatile solvents is a concern. Samples and reagent plates should be prepared immediately before running the protocol to minimize evaporation. AssayMAP Peptide Cleanup Protocol Guide 10

11 Preparing your reagents Introduction to the Peptide Cleanup Calculator The Peptide Cleanup Calculator is an Excel worksheet located on the Workflow Navigator that uses formulas to calculate reagent needs and recipe volumes. Based on the number of samples to process, the calculator manages dead volume, pipetting overage, and evaporation concerns for your experiment. The Calculator is comprised of two panels for 1. Calculator Data Entry and 2. Reagent Preparation. Using the Peptide Cleanup Calculator To use the calculator, you enter values into the green cells for the number of samples, for the volumes used in the Equilibrate, Cup Wash, Internal Cartridge Wash, Stringent Syringe Wash, Elute, and Re-Equilibrate steps, and for the number of wash cycles used in the Cup Wash and Stringent Syringe Wash steps. Acceptable range values are shown for each of these items. A formula calculates the number of plate columns used from the number of samples. Make reagents according to the recipes provided in the Reagent Preparation panel of the calculator. AssayMAP Peptide Cleanup Protocol Guide 11

12 Table Calculator Data Entry fields Field Range Default Definition, Instructions & Notes Samples Enter the number of samples you intend to run. Note: The number of samples should be a multiple of 8. Columns of Cartridges used This field is calculated based on the number of samples. This is the number of cartridge columns and reagent columns that will be prepared to manage samples. Equilibrate This field is the volume of utility buffer used to equilibrate the cartridges. Cup Wash This field is the volume of utility buffer used to wash out the cartridge cup and the syringes to ensure that no sample contamination remains in the cup This field is the number of times to perform the cup wash step and its accompanying syringe wash cycle. Internal Cartridge Wash Stringent Syringe Wash This field is the volume of utility buffer used to minimize non-target element contaminants from the internal parts of the cartridge This field is the volume of syringe wash buffer used to comprehensively clean the syringes immediately before elution to ensure there is no carryover This field is the number of times to perform the stringent syringe wash of the bare syringe probes. Elute This field is the volume of elution buffer used to elute peptides from the cartridges. Re- Equilibrate This field is the volume of utility buffer used to displace organic elution buffer and return the cartridge ph to a neutral condition. NOTE The cells where you can enter data provide color cues to identify how the data meets with expectations. Green: is a cell that requires you to enter a value. As long as the cell stays green, the value meets all of the requirements, go. Yellow: The value meets some of the requirements, but use caution. Red: The value does not meet the requirements, stop. AssayMAP Peptide Cleanup Protocol Guide 12

13 Preparing reagents for the Peptide Cleanup protocol The simplest Bravo deck configuration for the Peptide Cleanup protocol uses two solvents in addition to the deionized water used at the wash station. Table Reagent preparation Reagent Composition, Instructions & Notes Syringe Wash Buffer 50% ACN:50% H20:0.1% TFA * Because Stringent Syringe Buffer at deck location 5 is used for both syringe washing and priming, its composition should not be altered without evaluation of how the change could affect the priming step. Elution Buffer 50% ACN:50% H20:0.1% TFA * The typical composition of the Elution Buffer is the same as the Syringe Wash Buffer. Utility Buffer 100% H20:0.1% TFA * Due to the volume change that occurs when mixing organic and aqueous solutions, the actual prepared volumes (and v/v proportions) may not be exactly equal to the values indicated. The Peptide Cleanup calculator is designed to prepare enough extra volume to account for volume changes due to mixing effects. The Calculator takes into consideration that 96-well plates (PCR, round-bottom, and deep-well versions) require about μl of dead volume per well and a 12-channel reservoir plate requires about 3 ml of dead volume per channel. Reagents should be dispensed immediately before running the protocol to avoid evaporation 1 Pipette Syringe Wash Buffer, Elution Buffer, and Utility Buffer into its appropriate reservoir. a b c Pipette the specified volume of Syringe Wash Buffer into each channel being used in the reservoir plate to be placed at deck location 5. Pipette the specified volume of Elution Buffer into each channel being used in the reservoir plate to be placed at deck location 6. Pipette the specified volume of Utility Buffer into each channel being used in the reservoir plate to be placed at deck location 8. 2 If bubbles are present in the reagent plates, it may be necessary to centrifuge to remove the bubbles. NOTE If you are using fewer than 96 cartridges, ensure that you fill the labware columns, rows, or wells to correspond with the samples at deck location 4 and cartridge positions on the 96AM Cartridge & Tip Seating Station. AssayMAP Peptide Cleanup Protocol Guide 13

14 Setting up a Protocol Starting up 1 Ensure that the main power cable and Ethernet or serial cables are plugged into the connection panel. 2 Clear the deck except for the 96AM Wash Station (Location 1) 3 Toggle on ( ) power switch on the side of the AssayMAP Bravo. The green light on the switch illuminates when the AssayMAP Bravo Platform is on. 4 Turn on any accessories, for example, Pump Modules. 5 Turn on the computer and the monitor, and start the Microsoft Windows operating system. Installing software and opening the application interface The AssayMAP Peptide Cleanup protocol and app work with the VWorks software installed on the computer that runs the AssayMAP Bravo Platform. For more information about the VWorks software, see the VWorks Automation Control Setup Guide or contact Automation Solutions Technical Support. To install the Launch Pad, application interfaces, protocols, and utilities: 1 Navigate to the downloaded copy of the installer on your computer, and double-click the file AgilentAssayMAPLaunchPad_build exe 2 Follow the instructions that appear in the installation wizard window. 3 Click Finish. NOTE You can download the most recent version of the installer from NOTE Warning messages may appear asking you how to handle existing labware definitions, device profiles, and liquid classes. Follow the onscreen instructions. Opening the protocol and app To open the protocol and application interface from Launch Pad: 1 Open Launch Pad by double-clicking the AssayMAP Launch Pad shortcut on your desktop or from the Windows Start menu For XP: Programs\Agilent Technologies\AssayMAP Launch Pad For Win7: All Programs\Agilent Technologies\AssayMAP Launch Pad The Launch Pad appears. 2 Click the Peptide Sample Prep tab. AssayMAP Peptide Cleanup Protocol Guide 14

15 Figure Launch Pad web interface showing the Peptide Sample Prep options 3 Click any of the 4 icons (In-Solution Digestion, Peptide Cleanup, Fractionation, or Utilities) to Launch the Peptide Sample Prep: Workflow Navigator. The VWorks software launches and the User Authentication dialog appears. 4 Insert your User name and Password and click OK to login. The Peptide Sample Prep: Workflow Navigator appears inside a VWorks window. 5 All of the necessary tools and protocols are accessible from the Workflow Navigator. AssayMAP Peptide Cleanup Protocol Guide 15

16 Priming the wash station and initializing the Bravo platform Priming ensures that the tubing from the source bottle and the chimneys in the wash station are filled. Use the following procedure to prime the wash station and initialize the platform. For additional information about the Pump Module, tubing connections, and setup of source and waste bottles, see Pump Module User Guide. WARNING When you initialize the Bravo Platform, the robot head and tie bar can move. To prevent potential injury, keep clear of the device while it is in motion. To Initialize the Bravo: Click the Initialize button in the Workflow Navigator. 1 If this is the first action performed by the Bravo platform for the day, an initialization process automatically starts. 2 A series of messages will appear during the initialization process. NOTE A microplate-in-gripper error message appears, regardless of whether the gripper is or is not holding a plate. If the gripper is not holding labware, click Ignore and Continue, leaving device in current state to continue the homing process. If the gripper is holding labware, click Abort to cancel the initialization, remove the labware from the gripper, and restart by clicking Initialize All Devices. NOTE A home the W-axis error message appears during every initialization process to alert you that liquid may be in the syringes if a run was aborted immediately before the current run. If a fluid-in-tips message appears, but no fluid is in the tips, click Retry to continue homing the w-axis. If you suspect that there is fluid in the tips, click Ignore and Continue, leaving device in current state. This ensures that the liquid in the syringes is not dispensed from the Bravo head to a position on the deck. The AssayMAP Bravo will continue the initialization process until the head reaches the wash station. The W-axis is not homed error message shown below will appear. NOTE If you clicked Ignore and Continue, leaving device in current state as a response to the message shown above, the W-axis is not homed message appears: Click Diagnostics to enter the VWorks diagnostic tools, click Home W, Enable Motor, and then OK. This will dispense any liquid in the syringes into the wash station to waste, complete the initialization process as well as the prime and wash procedure. To prime the wash station: 1 Fill the 10 L source bottle for the 96AM Wash Station with purified deionized water, ensure the waste bottle is empty, and verify that the tubing connections are intact. AssayMAP Peptide Cleanup Protocol Guide 16

17 3 Make sure that there is no labware on the deck except for the wash station. 4 Click Prime and Wash. To verify that the Bravo Platform is ready: 1 Carefully observe the top of the chimneys during the Prime and Wash procedure. Fluid should flow out of the top of each chimney and the shape of the fluid dome that comes out the top of each chimney should be consistent. If fluid does not appear to be flowing out of a chimney, a bubble may be blocking the flow. In this case, click Prime and Wash to repeat the procedure. If any chimney has a fluid dome shape that is different than the others, carefully examine the top of the chimney for an irregular shape. Chimney irregularities can result from head crashes into the wash station, may result in large droplets clinging to the syringe probes, and can introduce variability in the assay. 2 Ensure that the indicator lights on the front of the Bravo Platform are blue, and that the robot-disable button on the pendant is activated (red indicator light). NOTE If you change the composition of the solution in the source bottle between runs, ensure that the tubing between it and the chimneys in the wash station fills with purified deionized water. In some instances, this may require disconnecting tubing connections, draining and refilling tubing lines, and reconnecting tubing. Figure Indicator light locations on the Bravo Platform Figure Activating the robot-disable button NOTE You only need to activate the robot-disable button after it has been used to disable a run. AssayMAP Peptide Cleanup Protocol Guide 17

18 Running a protocol 1 Prepare sample plates. a b c d Use the Single Liquid Addition Utility on the Workflow Navigator to adjust the buffer composition of the samples for reversed phase cleanup (i.e. change ph or reduce organic solvent concentration). (Optional) Use the Sample Plate Transfer Utility on the Workflow Navigator to move samples into one of the labware options available in the dropdown list for Deck Location 4 of the Labware Table. A µl sample excess in each well is recommended for optimal sample loading onto cartridges. Samples should be prepared immediately before running the protocol to avoid evaporation. Ensure the samples do not contain bubbles. (optional) Centrifuge to remove bubbles. 2 Use the Cartridge Transfer Utility to move cartridges to the Cartridge and Tip Seating Station. Click Cartridge Transfer in the Peptide Cleanup Banner of the Workflow Navigator and click Restore Defaults. Fill in Application Settings. You will use the Number of Columns of Cartridges from the Calculator as the Number of Columns to Transfer in the Application Settings. a b c Confirm that the physical Deck Layout exactly matches the Deck Layout diagram (B) and Labware Table (C) section of the app and that that each piece of labware seats properly on its plate pad. See the Utilities Guide in the Workflow Navigator for more detailed instructions on Cartridge Transfer Utility Click Run Cartridge Transfer. The head transfers cartridges from the Source: Cartridge Rack to the Destination: Seating Station at deck location 2. Click OK when the Protocol Complete message appears. d Remove unused cartridges from Deck Position 6. e Click Return to Navigator and open the Peptide Cleanup Protocol 3 Set up the Peptide Cleanup protocol. a (optional) Click Reset Defaults to set all Application Settings to their defaults. AssayMAP Peptide Cleanup Protocol Guide 18

19 b Enter the number of columns, choose the steps to perform ( )or skip ( ), and enter setting values. The Number of Full Columns of Cartridges must match the value from the Peptide Cleanup Calculator for Columns of Cartridges used Application Settings Number of Full Columns of Cartridges Steps Notes Set up samples in columns; cartridge layout and reagent plate layouts must match this configuration. Use value for Columns of Cartridges used from the Peptide Cleanup Calculator. Conduct Step? Default Value (Range) 1 (1-12) Volume (µl) Flow Rate (µl/min) Wash Cycles Initial Syringe Rinses syringes with deionized water in the specified number of cycles. 5 (1-10) Wash Prime Prime the cartridges with syringe wash buffer. Equilibrate Defines the volume of utility buffer used to equilibrate the cartridges. 50 (0-250) 25 ( ) 0 (0-10) Load Samples Defines the volume of sample, the flow rate used to load a sample and the 50 (0-245) 5 ( ) 5 (0-10) number of syringe wash cycles. Ensure that each sample well has an excess of at least µl to ensure that air does not get introduced into the cartridge. A 5-µL sample chase with utility buffer occurs after sample loading. Cup Wash Defines the volume of utility buffer used to wash out each cartridge cup 50 (1-100) 2 (0-10) and the number of times to perform the cup wash/syringe wash cycle. Internal Cartridge Wash Defines the volume of utility buffer, the flow rate used to wash non-target elements from the cartridge resin, and the number of syringe wash cycles. 50 (0-250) 25 ( ) 3 (0-10) Stringent Syringe Wash Elute Re-Equilibrate Final Syringe Wash Defines the volume of syringe wash buffer and number of wash cycles used to wash each syringe immediately before elution. 50 (1-250) 1 (1-10) Defines the volume of elution buffer, the flow rate used to elute peptides 25 (0-250) 5 ( ) 0 (1-10) from the cartridges and the number of syringe wash cycles. Defines the volume of utility buffer and the flow rate used to displace 50 (0-250) 25 ( ) elution buffer from the cartridge. Rinses syringes with deionized water in the specified number of cycles. 3 (0-10) c Place Sample Plate at deck location 4. d Place the other reagent plates at their indicated positions AssayMAP Peptide Cleanup Protocol Guide 19

20 e Confirm that the physical Deck Layout exactly matches the Deck Layout diagram (B) and Labware Table (C) section of the app and that that each piece of labware seats properly on its plate pad. To prevent head crashes, ALWAYS use the specified labware at its designated location and ensure that labware seats properly on its plate pad 4 Run the Peptide Cleanup protocol. Click Run and follow the progress of your experiment in the Control Panel status bar. When you see the Protocol Complete message appear, remove each Sample Plate from the deck. NOTE When the protocol starts, you can walk away from the Bravo Platform for the duration of the protocol. The default protocol with 50 μl samples and 5 μl/min flow rate should take about 35 minutes to complete. 5 Analyze results Pausing or aborting the run While conducting a protocol run or simulation, click Pause. The protocol pauses and the VWorks Scheduler Paused dialog box opens. NOTE While the Schedule Paused dialog box will appear, the protocol may not pause immediately. When you click Pause, the platform will complete the task currently running, which could take minutes to an hour to finish, depending on the volume and flow rate of the task. Generally, Load Samples is the longest step in the protocol. You will know that the pause starts when the head lifts up above the plate and stops. In the Scheduler Paused dialog box, click one of the following: Button Continue Description Resumes the protocol run when you are ready to continue. AssayMAP Peptide Cleanup Protocol Guide 20

21 Abort process Aborts the run. Select Abort process if you have determined that the protocol run is not recoverable. For details on the other options, see the VWorks Automation Control User Guide. Cleaning up at the end of the day Because the Peptide Cleanup protocol uses purified, deionized water in the source bottle, there are no special cleanup procedures for the liquid handling platform other than disposal of liquids from the Organic Waste plate. CAUTION Discard waste according to waste disposal procedures and in compliance with all applicable regulations and personal safety protection practices. Shutting down 1 Remove the labware from the deck. 2 Discard solutions and used labware following appropriate waste disposal procedures. 3 (optional) Use the Syringe Wash utility to perform a stringent syringe wash to minimize the risk of sample carryover into the next protocol. 4 Empty the Waste Bottle. 5 Close VWorks and, shut down the computer. 6 Turn off any accessories (Pump Modules and temperature controller). 7 If using an Auto Filling Reservoir, disconnect the bottles to prevent siphoning. 8 Toggle the power switch to the off (o) position. AssayMAP Peptide Cleanup Protocol Guide 21

22 Assay Development guidelines Setup and equilibration The first stage in the Peptide Cleanup protocol is to set up the system and prepare the cartridges for sample loading. This involves washing the syringes in the Bravo 96AM head to ensure there is no carryover from previous experiments, priming the cartridges to condition the resin and remove entrapped air, and equilibrating the cartridges to create optimal conditions for binding. Cartridges ship dry and therefore contain air entrapped in the cartridge bed. Failure to prime cartridges can prevent access of the sample and buffers to parts of the bed, resulting in reduced capacity and poor reproducibility. Initial Syringe Wash The Initial Syringe Wash step uses the wash station to flush any potential contaminants from the syringes in the head. During each Initial Syringe Wash cycle, the head aspirates 250 μl into the syringes from the wash station chimneys and then moves by a fixed offset between the chimneys to dispense to waste. Select the Initial Syringe Wash step to clean the syringes. Skip this option if you are certain that the syringes are clean. Setting the Initial Syringe Wash step parameters Parameter Value Notes Wash cycles 1 10 Default: 5 Increase the number of wash cycles to better clean the syringes. Note that increasing the number of cycles increases the time of the assay and consumption of reagents, and affects wear on the syringes. Prime Cartridges The syringes fill with sufficient syringe wash buffer (100 μl) to perform the Prime step. After mounting the cartridges on the probes, the head moves to the Organic Waste plate to prime the cartridges by dispensing 100 μl at 300 μl/min through the cartridges to waste. The last task of the priming step ejects the cartridges into the seating station. The priming solution (syringe wash buffer) requires the solution contain at least 25% organic solvent. Higher levels of organic are also acceptable and maybe preferred if large samples are to be loaded onto the cartridge to avoid changes in the solution s composition while it is sitting on the deck. When to select this option Always perform the Prime step at the beginning of a run. In the default protocol, this step is selected. AssayMAP Peptide Cleanup Protocol Guide 22

23 Equilibrate Cartridges The syringes aspirate the specified volume of utility buffer and then dispense at the specified flow rate through the cartridges into the wash station. The wash station washes the outside of the cartridges. The head moves to the seating station and ejects the cartridges. The head moves the syringes to the wash station at deck location 1 and washes the syringes with 250 µl of H2O for the specified number of cycles. When to select this option This step exchanges the buffer used for priming with utility buffer within the resin bed. It is critical to equilibrate the cartridge bed with a buffer that establishes optimal chemical conditions for sample loading. In the default protocol, this step is selected. Setting the Equilibration step parameters Parameter Value Notes Volume Flow rate μl Default: 50 μl μl/min Default: 25 μl/min The default volume for this step is equal to 10 column volumes, which should be sufficient for complete buffer exchange. A rate slower than 25 μl/min will likely have no benefit, but will increase the total assay time. A rate faster than 25 μl/min might not equilibrate through the pores in the beads across the full length of the cartridge bed. Wash cycles 0 10 cycles Default: 0 This allows you to control the number of syringe washes after the equilibration aspiration step by setting the value up to 10 wash cycles. The default setting is 0 cycle. Load Samples During Load Samples, the AssayMAP Bravo mounts the peptide cleanup cartridges onto the syringes, aspirates the specified volume of samples through the cartridges at the specified flow rate, and performs an external cartridge tip wash at the wash station, where cartridge tips dip into the wash station chimneys to remove any sample on the outside of the cartridge. AssayMAP cartridges can accommodate volumes ranging from 10 to 245 μl. After sample loading, a 5 µl chase volume of Utility Buffer will be automatically aspirated through the cartridges to ensure that the entire sample volume has been fully exposed to the entire resin bed. Following sample loading and chase the cartridges ejected into the tip seating station and then the are the flow through plus chase are collected into the Flow through plate (deck location#7) What is the binding capacity of the cartridge? For each AssayMAP peptide cleanup cartridge, the quantitative binding capacity depends on the relative hydrophobicity of the peptides in the sample, the sample complexity and the chemistry. As an example, the choice of ion pair reagents will affect binding characteristics. When examining human insulin in isolation, the quantitative binding capacity is approximately 400 µg per peptide cleanup cartridge. AssayMAP Peptide Cleanup Protocol Guide 23

24 When examining a tryptic digestion of BSA, the quantitative binding capacity is approximately 150 (without loss of hydrophilic peptides) to approximately 300 µg (with some loss of highly hydrophilic peptides) for this complex mixture. The total binding capacity (supersaturating amounts) is the maximum mass of peptide that can bind to the cartridge. If quantitative binding is not required, greater mass can be loaded. What is the concentration of the target in the sample? Use this formula to determine the volume of sample to load: μl sample to load = μg peptide desired μg/μl of peptide in sample When to select this option Always select the Load Samples step during a normal run. In the default protocol, this step is selected. Setting the Load Samples step parameters Parameter Value Notes Volume μl Default: 50 The volume depends on the concentration and the mass of sample to capture. It is not possible to aspirate all of the liquid from a microplate well. Add at least μl extra sample liquid to each well to ensure that the sample does not deplete fully, which would result in aspiration of air into the cartridges. Large sample volume or slow load rates may require slightly more overage. Flow rate μl/min Default: 5 μl/min The value depends on a balance between the speed of the assay and desired recovery. Binding capacity is inversely proportional to flow rate. For many resins, a flow rate of 20 μl/min results in a low dynamic binding capacity. It also produces some loss of total binding capacity, as the sample does not have sufficient time to diffuse throughout the pores in the cartridge resin bed. As the rate increases above approximately 20 μl/min, the total binding capacity continues to decrease until you hit a failure point when cavitation occurs in the cartridge, which happens only in aspiration mode. At this point, the liquid no longer draws through the cartridge. Generally, this failure occurs at approximately μl/min. If the flow rate is too slow, the protocol can run at non-optimal speed without significant additional binding. Wash cycles 0 10 Default: 5 Generally, the syringes should be washed as part of the Load Sample step. Under certain circumstances, you can skip this wash step to minimize run time. AssayMAP Peptide Cleanup Protocol Guide 24

25 Cup Wash The Cup Wash addresses the small amount of sample liquid that may remain above the upper cartridge membrane after sample loading. This step removes this residual liquid by filling the cartridge cups with utility buffer and then removing it along with the residual sample solution. You control the number of Cup Wash cycles by changing the number of wash cycles in the app. Each Cup Wash is followed by a syringe wash at the wash station. When to select this option Select this step if the presence of unbound sample liquid in the cartridge cups could cause complications with analysis. Internal Cartridge Wash The AssayMAP Bravo aspirates utility buffer through the cartridges, removes the cartridges from the probes, and dispenses the contents of the syringes in the wash station. The head then washes syringes at the wash station with 250 µl of deionized water. When to select this option Use Internal Cartridge Wash to minimize non-target elements from the sample matrix in the cartridge, which could possibly contaminate the elution. In the default protocol, this step is selected. Setting the Internal Cartridge Wash step parameters Parameter Value Notes Volume μl Default: 50 μl This allows the user to define the volume of utility buffer to aspirate through the cartridges during the internal cartridge washing process. Flow rate μl/min Default: 25 μl/min A rate slower than 25 μl/min will likely have no benefit, but will increase the total assay time. A rate faster than 25 μl/min might not equilibrate through the pores in the beads across the full length of the cartridge bed, which leads to incomplete washing. Wash cycles 0 10 Default: 3 This parameter specifies the number of syringe wash cycles performed after an Internal Cartridge Wash step at the wash station. Stringent Syringe Wash This syringe wash step comprehensively cleans the syringes with the syringe wash buffer to ensure there is no carryover. Use the Wash Cycles setting for this step to control the number of times this step repeats. When to select this option Use a Stringent Syringe Wash when rigorous cleaning of the syringes is necessary to prevent carryover. In the default protocol, this step is selected. AssayMAP Peptide Cleanup Protocol Guide 25

26 Setting the Stringent Syringe Wash step parameters Parameter Value Notes Volume μl Default: 50 μl This allows you to define the volume to aspirate from the Stringent Syringe Wash and dispense through the syringes to Organic Waste. Wash cycles 1 10 Default: 1 This parameter specifies the number of syringe wash cycles performed with elution buffer. Elute The AssayMAP Bravo aspirates elution buffer using the bare probes. After mounting the cartridges, the head dispenses elution buffer through the mounted cartridges into the Eluate Collection plate. Under optional elution conditions, peptides fully elute 2 column volumes (10 ul). When to select this option Always select Elute to remove bound peptide from cartridges in a normal run. In the default protocol, this step is selected. Setting the Elute step parameters Parameter Value Notes Volume μl Default: 25 μl This allows you to define the volume of elution buffer aspirated into the syringe from deck location 6 and dispensed through the cartridges for Eluate Collection at deck location 9. Flow Rate μl/min Default: 5 μl/min A flow rate slower than 10 µl/min is unlikely to improve the elution yield. There is very little difference in yield using a flow rate from 2 to 20 µl/min Wash cycles 1 10 Default: 0 This parameter specifies the number of syringe wash cycles performed with elution buffer. Final Syringe Wash The Final Syringe Wash uses deionized water from the wash station to flush any potential contaminants from the syringes. The head performs these washes on the wash station. The AssayMAP Bravo aspirates 250 μl of fresh deionized water into the syringes using the bare probes and dispenses at an offset between the chimneys on the wash station. When to select this option Select Final Syringe Wash after each run to clean the syringes thoroughly before running a different assay. In the default protocol, this step is selected. AssayMAP Peptide Cleanup Protocol Guide 26

27 Setting the Final Syringe Wash step parameters Parameter Value Notes Wash cycles 1 10 Default: 3 Increase the number of wash cycles to better clean the syringes. Note that increasing the cycle number increases the duration of the assay and consumption of reagents, and affects wear on the syringes. NOTE The amount of carryover in the syringe (expressed as dilution of the starting sample) depends on the total number of syringe wash cycles. Five cycles give a dilution of up to In cases where carryover is a major concern, increasing the number of iterations also increases the dilution factor, but with a cost of increased assay time and reduced syringe lifetime. AssayMAP Peptide Cleanup Protocol Guide 27

28 Automation Protocol Steps This section describes the basic movements of the automation when a default protocol is selected. Changing the selections or deselecting options will alter the movement. Head moves to deck position: Action: Cartridge Transfer Protocol 6 Load the desired number of cartridges 2 Park cartridges on the Cartridge & Tip Seating Station and adjust their orientation Peptide Sample Cleanup Protocol 2 Head check Initial Syringe Wash 1 Initial Syringe Wash, repeat for the number of cycles Prime 5 Aspirate Syringe Wash for Prime 2 Load cartridges 3 Dispense Syringe Wash through cartridges into Organic Waste for priming step 2 Park cartridges 1 Wash Syringes Equilibrate 8 Aspirate Utility Buffer for equilibration 2 Load cartridges 1 Dispense Utility buffer through cartridges to equilibrate 2 Park Cartridge 1 Wash syringes, repeat for the number of cycles (default is 0) Load Samples 2 Load cartridges 4 Aspirate samples to load onto cartridges 1 External Cartridge Wash 8 Aspirate Utility buffer for sample chase 2 Park cartridges 7 Dispense flow through plus chase into flow through collection plate 1 Wash syringes, repeat for the number of cycles Cup Wash 8 Aspirate Utility buffer 2 Perform Cup Wash 1 Wash syringes, repeat for the number of cycles Internal Cartridge Wash 2 Load cartridges 8 Aspirate Utility buffer into cartridges for Internal Cartridge Wash 1 Dispense Internal Cartridge Wash 2 Park cartridges 1 Wash syringes, repeat for the number of cycles Stringent Syringe Wash 5 Aspirate Syringe Wash Buffer 3 Dispense into Organic Waste, repeat for the number of cycles 1 Wash syringes, repeat for the number of cycles AssayMAP Peptide Cleanup Protocol Guide 28

29 Elute 6 Aspirate Elution Buffer 1 Wash Syringe tips 2 Load cartridges 9 Elute Samples into Elution Collection Plate 1 Wash external cartridge 2 Park Cartridges Re-Equilibration 8 Aspirate Utility Buffer for re-equilibration 2 Load cartridges 1 Dispense Utility buffer through cartridges into wash station 2 Park cartridges Final Syringe Wash 1 Final Syringe Wash syringes, repeat for the number of cycles AssayMAP Peptide Cleanup Protocol Guide 29