Overview: How to make sample preparation for LC-MS analysis easy

Downloads: App note and protocols for sample preparation for LC-MS analysis with the ASSIST PLUS pipetting robot

Experimental set-up

Experience reproducible results while having your hands free by using our easy sample preparation protocols for peptides, including high volume dilutions for LC-MS analysis (Figure 1).

Step-by-step procedure

1. Dissolve samples in DMSO 

Dissolve freeze-dried peptides in 100 % DMSO

Place a 25 ml multichannel reagent reservoir filled with 10 ml of 100 % DMSO on deck position A (Figure 2, blue), and an INTEGRA rack with 1.5 ml microcentrifuge tubes containing freeze-dried peptides on deck position B (Figure 2, pink).

Select and run the VIALAB program ‘Dissolve_samples_in_DMSO'. With 1250 µl low retention, sterile, filter GRIPTIPS, the 8 channel 1250 µl VOYAGER adjustable tip spacing pipette aspirates 1 ml of DMSO from the reservoir (Figure 3a) and dispenses it into the 1.5 ml centrifuge tubes on deck position B (Figure 3b). Mixing 10 times at a gentle speed (6) ensures a homogeneous solution of peptides.

Tips:

• Easy adjustment of mixing parameters in VIALAB helps mix reagents of different solubilities.
• Accurate pipetting of DMSO with low retention GRIPTIPS and a reduced pipetting speed.

2. Dilute samples in methanol

Worklist set-up for individual peptide dilutions

.csv files are created using the VIALAB worklist template (Figure 4) or imported directly into the VIALAB worklist step. The first worklist step includes the worklist for diluent distribution to analyze eight peptides in triplicates, and the second worklist step, the worklist for the different peptides.

Tips:

• Worry-free exchange of worklists for different sample dilutions without changing the VIALAB program.
• Quick adaption to varying sample counts when deselecting samples in the worklist step.

Sample dilution with methanol in triplicates

A 25 ml multichannel reagent reservoir filled with 25 ml of 100 % methanol is placed on deck position A (Figure 5, blue), and dissolved peptides in 1.5 ml tubes with an INTEGRA rack on deck position B (Figure 5, pink). An INTEGRA rack with 24 empty HPLC vials is placed on deck position C (Figure 5, green).

Select and run the VIALAB program ‘Dilute_samples_with_methanol’. The D-ONE single channel pipetting module on the ASSIST PLUS pipetting robot with 1250 µl sterile, filter GRIPTIPS distributes diluent from position A to position C (Figure 6a) as indicated in ‘worklist_diluent’. An initial tip pre-wetting prevents dripping when pipetting volatile liquids by saturating GRIPTIPS with vapor. Decreasing the speed to 5 with a one second delay after pipetting followed by a tip touch against the side of the HPLC vial, increases accuracy when dispensing methanol. The D-ONE then individually dilutes peptides from position B to position C (Figure 6b) in triplicates using 125 µl low retention, sterile, filter GRIPTIPS, as indicated in ‘worklist_samples’. Every peptide is mixed five times with the full 125 µl GRIPTIP volume.

Tips:

• Worry-free use of different sample aliquots with the D-ONE's LLD function.
• Avoid dripping caused by vibrations with reduced movement speeds during pipette travel.
• Risk-free methanol pipetting because the ASSIST PLUS fits easily in a fume hood.

Remarks

• Low volume HPLC vials: Special low volume HPLC vials with inlets are compatible with the INTEGRA rack for HPLC vials and accessible by the D-ONE (Figure 7).
• VIALAB software: The VIALAB programs can be easily adapted to your specific labware and protocols, for instance when partial plates are needed.
• Partial plates: Programs can be adapted at any time to a different number of samples, giving laboratories total flexibility to meet current and future demands.