Preparing the reagents

 

Make sure you filter salt-containing buffers if salt precipitation is a risk. Salt precipitates can clog cartridges and cause poor performance.

 

 

A build-up of salts within the syringe barrels can corrode the syringe seals. Therefore, you should filter salt-containing buffers before use. In addition, you should use the System Shutdown utility to clean the syringes after every protocol run.

 

The following tables lists the recommended buffers to use as a starting point for optimizing the phosphopeptide enrichment chemistry. The simplest AssayMAP Bravo deck configuration for the default Phosphopeptide Enrichment protocol uses three solutions in addition to the deionized water used for washing syringes at the wash station.

 

Reagent	Deck location and composition
Priming Buffer and Syringe Wash Buffer	Deck location 5 Fe(III)-NTA: 99.9% ACN : 0.1% TFA TiO2: 50% ACN : 45% H2O : 5% NH3 Because this dual-purpose buffer serves as both the Priming Buffer and Syringe Wash Buffer during the protocol run, do not alter the composition without evaluating how the change affects the priming step. Organic solvent in the buffer helps to purge entrained air within the cartridge resin bed during priming and is effective at removing hydrophobic species from syringes as part of the stringent syringe wash. Priming with a high-pH buffer helps to better prepare TiO2 cartridges than priming with a low-pH buffer.
Elution Buffer	Deck location 6 Fe(III)-NTA: 99 % H2O : 1% NH3 TiO2: 80 % H2O : 15% ACN : 5% NH3 Dilute solutions of aqueous ammonia are sufficient to elute phosphopeptides from either cartridge type. Recovery of hydrophobic peptides may be improved with the addition of small percentages of acetonitrile.
Equilibration and Cartridge Wash Buffer	Deck location 8 Fe(III)-NTA: 80% ACN : 19.9% H2O : 0.1% TFA TiO2: 50% ACN : 48 % H2O : 2% TFA The composition of this reagent may match that used for the sample loading buffer.

Note: All suggested solutions listed as percentages are volume/volume formulations.

Note: The source of NH3 for these buffers is aqueous ammonium hydroxide, which is commonly listed as %NH3 in H2O from commercial suppliers.

 

A small volume excess is required in all labware types to ensure proper volume transfer. Use the Reagent Volume Calculator to automatically include excess volume, or to look up recommended values for each allowable labware type.

 

Note: You can also find the recommended excess volume for each allowable labware type in the Labware Reference Guide, which is available in the Literature Library page of the Protein Sample Prep Workbench.

About the Reagent Volume Calculator for Phosphopeptide Enrichment

The Reagent Volume Calculator is a Microsoft Excel file that you access from the Protein Sample Prep Workbench. The calculator uses formulas to calculate estimated volume requirements for samples and buffers. Based on the number of samples to process, the calculator manages the dead volume, pipetting overage, and evaporation concerns for your experiment. For volume overage by labware type, see Labware options and excess volume recommendations.

The following figure shows the worksheet of the Reagent Volume Calculator for Phosphopeptide Enrichment.

Figure. Phosphopeptide Enrichment Calculator worksheet

 

 

Return to top

Using the Reagent Volume Calculator for Phosphopeptide Enrichment

To use the Reagent Volume Calculator:

1	Open the App Library. See Accessing an application.

2	Locate Phosphopeptide Enrichment, and then click Calculator. Microsoft Excel starts and displays the calculator.

3	Ensure that you enable content in Microsoft Excel.

4	In the calculator, enter the values in the green boxes (data entry fields):

–	Type the Number of Columns corresponding to the number of columns of AssayMAP cartridges to be used.

–	Type the volumes to use in the Prime, Equilibrate, Cup Wash, Internal Cartridge Wash, Stringent Syringe Wash, Elute and Eluate Discard steps, as well as the value for the Existing Collection Volume.

–	Select whether or not flow-through will be collected for the applicable steps.

See the following table for a description of each field, the default values, and the range of acceptable values.

5	Select the labware that will be used. The calculator will estimate the volumes required per well, channel, or reservoir including the volume of bulk solution required to fill the labware.

Note: The boxes where you can enter data provide color cues to identify how the data meets with expectations:

•	Green indicates a field where you enter a value. If the box stays green after you enter the value, the value meets all the requirements.

•	Yellow indicates a caution that the value meets only some of the requirements. Use caution in proceeding.

•	Red indicates a warning that the value does not meet the requirements.

If a box turns yellow (caution) or red (warning), flagging a potential issue, see the relevant Field description and value range in the table.

Table Calculator data entry fields

 

Field	Description
Number of Columns	The number of full cartridge columns in the 96AM Cartridge & Tip Seating Station and the corresponding number of reagent columns that will be prepared to manage samples. IMPORTANT Full columns of cartridges are required to ensure proper cartridge seating on the Bravo 96AM Head. Note: One full cartridge column consists of 8 cartridges. Value: • Default: 1 • Range: 1–12
Prime	The volume (µL) of Priming Buffer at deck location 5 used to prime and properly wet the cartridge resin bed. Value: • Default: 100 • Range: 0–250
Equilibrate	The volume (µL) of Equilibration Buffer at deck location 8 used to equilibrate the cartridge resin bed. Value: • Default: 50 • Range: 0–250
Load Samples	The volume (µL) of sample at deck location 4 that will be dispensed through the cartridge resin bed. Value: • Default: 100 • Range: 0–1000 The default option is to collect the flow-through from this step in the Flow Through Collection plate.
Cup Wash	The volume (µL) of Cartridge Wash Buffer at deck location 8 used to wash out the cartridge cup and the syringes to ensure that no sample contamination remains in the cup. Value: • Default: 25 • Range: 0–100
	The number of times to perform the cup wash step and its accompanying syringe wash cycle. Value: • Default: 1 • Range: 1–10
Internal Cartridge Wash	The volume (µL) of Cartridge Wash Buffer at deck location 8 dispensed through cartridges to minimize non-target element contaminants from the cartridge resin bed. Value: • Default: 50 • Range: 0–250 The default option is to not collect the flow-through from this step in the Flow Through Collection plate
Stringent Syringe Wash	The volume (µL) of Syringe Wash Buffer at deck location 5 used to comprehensively clean the syringes immediately before elution to reduce potential carryover. Value: • Default: 50 • Range: 0–250
	The number of times to perform the stringent syringe wash of the bare syringe probes. Value: • Default: 1 • Range: 1–10
Elute	The volume of Elution Buffer at deck location 6 that is used to elute phosphopeptides from the cartridge resin bed. Value: • Default: 20 • Range: 0–250
Eluate Discard	The volume (µL) of eluate to discard before collecting eluate in the Eluate Collection plate. Value: • Default: 0 • Range: 0–25 The default option is to not collect the flow-through from this step in the Flow Through Collection plate.
Existing Collection Volume	The volume (µL) of buffer added to the Eluate Collection plate before the start of the protocol to aid in recovery of small elution volumes or to immediately dilute eluate. Value: • Default: 0 • Range: 0–300

 

Return to top

Dispensing the reagents

 

To minimize evaporation, fill the labware immediately before run time or keep them covered until you run the protocol.

 

Before you start, make sure you have the following:

•	Three of the reservoir labware that are acceptable for the protocol. See Labware options and deck locations.

•	The reagent volume information from the calculator worksheet. See Using the Reagent Volume Calculator for Phosphopeptide Enrichment.

To dispense the calculated volumes of reagents into the reservoirs:

1	Label each reservoir labware to identify its contents:

•	Priming and Syringe Wash Buffer

•	Elution Buffer

•	Equilibration and Cartridge Wash Buffer

2	Dispense the buffers into their appropriate 96-well plate, channel plate, or reservoir:

a	Add the specified volume of Priming and Syringe Wash Buffer into the priming and syringe wash buffer plate or reservoir to be placed at deck location 5. Fill only the channels or wells that will be used.

b	Add the specified volume of Elution Buffer into the elution buffer plate or reservoir to be placed at deck location 6. Fill only the channels or wells that will be used.

c	Add the specified volume of Equilibration and Cartridge Wash Buffer into the equilibration and cartridge wash buffer plate or reservoir to be placed at deck location 8. Fill only the channels or wells that will be used.

3	If necessary, centrifuge the reagent labware to remove bubbles.

 

If you are using fewer than 96 cartridges, make sure you fill the labware columns, rows, or wells to correspond with the sample layout in the sample plate at deck location 4 and the arrange the cartridge positions in the 96AM Cartridge & Tip Seating Station to match. For more information, see Preparing the samples.

 

 

Return to top