Before you start

This topic lists the required hardware, software, labware, and reagents for running the Phosphopeptide Enrichment v2.1 protocol. If you have questions about these items, contact Agilent Customer Service.

Hardware

Required hardware

 

 

The following figure and table show the components of the AssayMAP Bravo Platform, which is required for running the AssayMAP protocols.

Figure. AssayMAP Bravo Platform components

 

 

Item	Required hardware
1	Gripper upgrade
2	Bravo 96AM Head
3	96AM Wash Station or the later model 96 Channel Wash Station
4	Pump Module 2.0 and two carboys
5	96AM Cartridge & Tip Seating Station
6	Risers, 146 mm
7	STC controller
8	Peltier Thermal Station with custom plate nest
9	Thermal plate insert
10	Orbital Shaking Station with Control Unit

 

Optional equipment

You might need the following when preparing the samples and reagents:

•	Microplate centrifuge, such as the Agilent Microplate Centrifuge or equivalent

•	Microplate vacuum concentrator for concentration and drying of samples

•	Microplate sealer, such as the Agilent PlateLoc Thermal Microplate Sealer or equivalent

 

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Software

 

 

The following table lists the minimum software requirements:

 

Software	Version
Agilent VWorks Automation Control	13.1.5
Agilent Protein Sample Prep Workbench	3.2
Microsoft Excel Required for the reagent volume calculators and method setup tools.	2016

 

 

 

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AssayMAP cartridges

The following table lists the cartridges for the Phosphopeptide Enrichment application. Each cartridge type can be purchased as a rack of 96 cartridges.

 

Cartridge type	Part number
AssayMAP Fe(III)-NTA cartridge rack	G5496-60085
AssayMAP TiO2 cartridge rack	G5496-60016
AssayMAP Resin-Free cartridge rack This cartridge can be used for mock runs or to fill remaining positions in columns that are partially occupied by Fe(III)-NTA or TiO2 cartridges, if necessary. For more information, see Preparing the samples.	G5496-60009

Cartridge use and storage guidelines

See the cartridge box label for guidelines for storing unused cartridges in the original shipping package.

Follow these guidelines to get the best performance from AssayMAP cartridges:

•	Use only particulate-free samples. Such samples can be from a previous chromatographic run or from samples that have been filtered through a 0.22-µm filter.

•	Use only particulate-free buffers and reagents. Ensure that salt-containing solutions and solutions prone to precipitation are filtered through a 0.22‑µm filter.

•	Use only primed and equilibrated cartridges.

 

Cartridges ship dry and, therefore, contain air entrained in the cartridge bed. Failure to prime the cartridges can prevent the reagents and buffers from accessing parts of the resin bed, resulting in reduced capacity and poor reproducibility.

 

•	Do not allow wetted cartridges to dry out.

Note: Cartridges will not dry out during the course of a normal application run. Cartridges can dry out rapidly if they are exposed to air for extended periods (e.g., >1 hour) after they have been primed and equilibrated.

If you need to store primed and equilibrated cartridges for a short period, ensure that you use the lidded blue rack-receiver plate stack with an appropriate solution in the receiver plate chimneys such that the cartridge tips are submerged in the solution.

•	AssayMAP cartridges are intended to be single-use consumables.

•	AssayMAP TiO2 cartridges are stable from pH 1 to 14, approximately.

•

AssayMAP Fe(III)-NTA cartridges are stable from pH 2 to 11, approximately. At levels > pH 3.5, approximately, resin within the cartridges may turn from pale yellow to golden yellow to orange or brown due to the formation of iron(III) complexes (most commonly with hydroxide). Resin coloration and intensity are a function of both the pH and the chemical nature of the solutions passed through the cartridge. Bare NTA cartridges are stable from pH 2 to 14, approximately.

•	AssayMAP Fe(III)-NTA cartridges that have been stripped (bare NTA cartridges) should be charged and used immediately.

Bare NTA cartridges that have been prepared properly may be stored at 4 °C to 8 °C for less than 1 week.

To prepare the cartridges for short-term storage, flush the cartridges with 20% ethanol and place them in the 96AM Cartridge Rack and Receiver Plate with 150 µL of 20% ethanol in each well.

 

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Labware options and deck locations

The following table lists all labware options for the protocol and the deck locations at which the labware are permitted. The accompanying figure shows the labware locations on the AssayMAP Bravo deck.

 

Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

 

Figure. Labware locations on the AssayMAP Bravo deck (top view)

 

 

Labware	Manufacturer part number*	Deck location options
12 Column, Low Profile Reservoir, Natural PP	Agilent 201280-100	3, 5, 6, 7, 8
8 Row, Low Profile Reservoir, Natural PP	Agilent 201282-100	3, 5, 6, 7, 8
96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom	ABgene AB-1127	3, 4, 5, 6, 7, 8
96 Eppendorf 30129300, PCR, Full Skirt, PolyPro	Eppendorf 30129300	4, 7, 9
96 Greiner 652270, PCR, Full Skirt, PolyPro	Greiner 652270	4, 7, 9
96 Bio-Rad PCR, Hard-Shell, Low-Profile, Full Skirt	Bio-Rad HSP‑9611	4, 7, 9
96 Greiner 650201_U-Bottom, Clear PolyPro	Greiner 650201	3, 4, 5, 6, 7, 8, 9
96 Greiner 650207_U-Bottom, White PolyPro	Greiner 650207	3, 4, 5, 6, 7, 8, 9
96 Greiner 651201_V-Bottom, Clear PolyPro	Greiner 651201	3, 4, 5, 6, 7, 8, 9
96 Costar 3363, PP Conical Bottom	Corning Costar 3363	3, 4, 5, 6, 7, 8, 9
96 Greiner 675801, Half Area, Flat-Bottom, UV Star	Greiner 675801	4, 7, 9
96 V11 Manual Fill Reservoir	Agilent G5498B#049	5, 6, 8
Reservoir, Axygen Scientific RES-SW96-LP, 86mL pyramid bottom	Axygen Scientific RES‑SW96-LP	5, 6, 8
Reservoir, Seahorse 201254-100, PP, no walls, pyramid bottoms	Agilent 201254-100	5, 6, 8
*For dimensionally equivalent alternatives and other labware details, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

 

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Labware options and excess volume recommendations

 

A small reagent volume excess is required in all labware types to ensure proper volume transfer. Use the Reagent Volume Calculator to automatically include excess volume, or refer to the following table for your labware type.

 

The following table provides recommended volume overages of aqueous solutions for the default protocol settings. The Phosphopeptide Enrichment Calculator automatically calculates the recommended excess reagent and sample volume for your labware selection. For details, see Using the Reagent Volume Calculator for Phosphopeptide Enrichment.

Table Excess volume recommendations by labware (for aqueous solutions using the default protocol settings)

 

Labware	Mfr part number	Excess volume (µL) per well, column, or reservoir
12-Column Low-Profile Reservoir	Seahorse 201280-100	3,000
8-Row Low-Profile PolyPro Reservoir	Seahorse 201282-100	4,500
1.2 mL Deep-Well PolyPro Clear Plate	ABgene AB-1127	30
96-Well Full Skirt PolyPro PCR Plate	Eppendorf 30129300	10
96-Well Full Skirt PolyPro PCR Plate	Greiner 652270	10
96-Well Full Skirt PolyPro PCR Plates	Bio-Rad HSP-9XXX	10
96-Well U-Bottom PolyPro Clear Plate	Greiner 650201	20
96-Well U-Bottom PolyPro White Plate	Greiner 650207	20
96-Well V-Bottom PolyPro Clear Plate	Greiner 651201	20
96-Well V-Bottom PolyPro Clear Plate	Corning Costar 3363	20
96-Well Flat-Bottom Half Area, UV Plate	Greiner 675801	25
96 V11 Manual Fill Reservoir	Agilent G5498B#049	20,000
PolyPro Reservoir, Pyramid Bottom	Axygen Scientific RES‑SW96-LP	20,000
PolyPro Reservoir, Pyramid Bottom	Seahorse 201254-100	20,000
Note: The recommended overages are only for aqueous solutions considering the length of time required to run the default protocol (~50 minutes). If volatile solvents are used or if the length of the protocol is increased, a larger excess volume may be required.

 

To prevent evaporation, reagents and samples should be prepared immediately before running the Phosphopeptide Enrichment protocol.

 

For dimensionally equivalent alternatives and other labware details, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

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Reagents

The volume, type, and concentration of reagents and buffers required for phosphopeptide enrichment will vary depending on the specific AssayMAP phosphopeptide enrichment cartridge being used, the sample characteristics, and the desired analytical result. See the Reference library for phosphopeptide enrichment by IMAC or TiO2 for literature with specific chemistry examples.

The simplest deck configuration for the Phosphopeptide Enrichment protocol has three buffers on the AssayMAP Bravo deck that are tailored to the sample and the desired experimental outcome. Typically, these buffers comprise the following:

 

Solution	Quality
Acetonitrile (ACN)	HPLC-grade
Trifluoroacetic acid (TFA)	HPLC-grade
Ammonium hydroxide (NH4OH)	HPLC-grade
Purified deionized water (H2O)	HPLC-grade, MilliQ, or equivalent

 

Use the provided Phosphopeptide Enrichment Calculator to determine volumes and suggested overages for the selected labware and specific protocol conditions. See Preparing the reagents.

 

 

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