Matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) with robot spotter

MS Index

LC-MS

Tandem MS

MALDI-MS

GC-MS

The Micromass TofSpec-2E™

MALDI-TOF-MS is the workhorse for proteomics. For organisms whose genome sequence is known the identification of 'interesting' protein spots from a 2-D gel is routine. Protein spots are excised and digested with a specific protease (e.g., trypsin cleaves only on the carbonyl side of lysine and arginine) and the fragments measured by MALDI-TOF. This peptide mass fingerprint can then be used to search databases to identify the protein (Blackstock & Weir, 1999; Yates, 2000).

In this MS method a solid matrix is used, which absorbs light at the wavelength the laser produces (we have both a Nitrogen UV (337 nm) and an Erbium:YAG IR (2940 nm) laser). The sample is mixed with a matrix solution and allowed to co-crystallise on a target plate. When the laser is fired at the target the matrix absorbs the laser light energy which vaporizes it (it desorbs from the surface) and this carries some of the sample with it. At the time that the laser is pulsed a voltage is applied to the target plate to accelerate the ionised sample towards a time-of-flight mass analyser. The Tof can be operated in either linear or reflectron mode and can easily separate proteins up to 500 kDa. The precision is considered to be approx 10 ppm.

The Micromass 2700MS™ Robot spotter

The preparation of samples can be really quite tedious and laborious, particularly with the very low volumes (1 µl) that are used in MALDI-TOF-MS. Therefore we use a robot to mix the sample with the appropriate matrix and then spot this mixture onto a MALDI target plate. This high throughput sample preparation method is robust, reproducible and allows a 96 target plate to be spotted in a few minutes.

References

·         Blackstock, W. P. & Weir, M. P. (1999). Proteomics: quantitative and physical mapping of cellular proteins. Trends in Biotechnolgy 17, 121-127.

Page maintained by Miss Charly Morgan
last updated 17th May 2004