A new application note from Agilent Automation Solutions describes a protocol for automated sample preparation and MALDI spotting
The protocol was developed with the protein analytics group of Dr Lottspeich at the Max Planck Institute of Biochemistry in Martinsried, Germany.
It automates the sample-preparation steps (cleansing and desalting) of peptides designated for mass spectrometry and the subsequent spotting onto a MALDI target plate using the Agilent Bravo Automated Liquid Handling Platform and C18 filled Velotips from Glygen.
In order to get high-quality peptide mass fingerprints in MALDI mass spectrometry, peptides must be concentrated and cleansed from impurities of previous steps.
In particular, salts and buffer components originating from the protein digests should be removed.
There are several manual cleansing tools available.
All are based on various chromatography media and require multiple aspiration/dispensing or mixing steps.
While manual sample handling remains a tedious procedure and extremely difficult to standardise, according to Agilent, automation of the routine has never been truly feasible on any automation platform due to several technical limitations.
In the application note, the authors compare automated sample preparation for MALDI-MS analysis on the Agilent Bravo Automated Liquid Handling Platform with a traditional manual cleansing process using Millipore Ziptips.
A protocol for the binding, washing, elution and direct spotting onto MALDI targets was created using Agilent Vworks.
Automation Control software is described that can be adapted to various MALDI target plates, labware and pipetting tasks.
Results show that peptide mass fingerprints produced by the automatic workflow performed equally well or better than the manual process regarding signal intensity and protein sequence coverage.
As an additional benefit, the whole process is carried out in a reproducible and timesaving manner.
The outcome of the experimentation shows that the automatic workup results in a better sequence coverage and Mascot score than both the tested manual workups.
The authors conclude that the Bravo Automated Liquid Handling Platform has been proven to be an effective tool for fully automated sample preparation with C18 Velotips and subsequent MALDI target spotting processes.
