Make sure the samples are free of macromolecular particulates, such as large protein aggregates and cellular debris to prevent clogging the cartridges. Samples should be filtered through a 0.45‑µm filter or centrifuged at a high g‑force before loading on an AssayMAP cartridge.

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 for standard, low-pH peptide cleanup with C18 or RP-S cartridges or basic 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 can acidify samples for low-pH peptide cleanup before loading onto AssayMAP C18 and RP-S cartridges generally by adding TFA, formic acid, or acetic acid. Sample pH for high-pH peptide cleanup is commonly adjusted using aqueous solutions of ammonium formate or ammonium acetate titrated to a pH > 10 using ammonium hydroxide. You can use the Reagent Transfer utility to perform these pH adjustments. For instructions, see Reagent Transfer v3.0 User Guide.

Samples containing organic solvents or some types of detergents should be avoided as they might 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 Reagent Transfer utility to modify the composition of your samples with a dilution or pH adjustment, especially after using the In-Solution Digestion workflow.

The AssayMAP Peptide Cleanup protocol permits loading up to 1000 µL of sample onto AssayMAP cartridges. For sample volumes > 250 µL, the protocol will iteratively load samples onto cartridges to stay within the maximum syringe volume (250 µL) of the Bravo 96AM Head.

Two ways to express the binding capacity of a cartridge are quantitative binding capacity and total binding capacity:

See the Agilent AssayMAP Bravo Cartridges Selection Guide for detailed information about the binding capacity for the 5 and 25 µL RPS and C18 cartridges.

If you know the approximate concentration of the target molecule in your sample and you are working within the quantitative binding range of the cartridge, you can determine the volume of sample to load as follows:

Before transferring the samples, you should plan the layout of the samples in the microplate. Consider the following:

See Labware for acceptable labware at each deck location.

An excess (overage) volume ensures that a microplate well or column does not fully deplete, which would result in aspiration of air into the syringes and then into the cartridges, compromising performance.

The Reagent Volume Calculator shows the recommended overage for the labware types being used and automatically includes recommended overages in the volume it recommends per well.

Labware-specific overage recommendations are also presented in the Labware Reference Guide, which you can find in the Literature Library page of the Protein Sample Prep Workbench. More or less overage can be used depending on the volatility of the solution and the length of the run but the recommended overages are fine for most standard runs.