No image problem for crabs: Coupling multidimensional separations with MALDI-MS imaging

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  • Published: Aug 20, 2012
  • Author: Jon Evans
  • Channels: Detectors
thumbnail image: No image problem for crabs: Coupling multidimensional separations with MALDI-MS imaging

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No image problem for crabs: Coupling multidimensional separations with MALDI-MS imaging

For the first time ever, US scientists have coupled multidimensional separation with mass spectrometry imaging (MSI), producing a system that is particularly effective at studying complex samples containing analytes at low concentrations.

The system is made up of liquid chromatography (LC) as the first dimension and capillary electrophoresis (CE) as the second dimension, followed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). As an MSI technique, MALDI-MS is usually used to analyze whole biological samples, such as bodily tissue, producing a spectral image that preserves the physical structure of the sample.

It involves placing the sample on a metal plate and then covering it with a liquid or solid matrix, often a compound called 2,5-dihydroxybenzoic acid (DHB). Scanning the sample with a laser beam causes the matrix molecules to vibrate rapidly, transforming the individual biomolecules within the sample into ions that can then be analyzed by time-of-flight MS.


Groovy slide

Because of the need to combine the sample with the matrix, MALDI-MS is not inherently compatible with separation techniques such as LC and CE. And as there are numerous forms of MS that are perfectly compatible, such as electrospray ionization (ESI) MS, this raises the question of why anyone would want to try coupling LC or CE with MALDI-MS. Well, because you can do things with MALDI-MS that you can’t do with other forms of MS.

‘The coupling of MSI enables continuous collection of LC or CE flow as an imaging trace, which greatly enhances the resolution from the separation dimension,’ explains Lingjun Li at the University of Wisconsin, Madison. ‘Furthermore, because CE or LC eluent is continuously deposited on a MALDI plate along with MALDI matrix, the samples can be archived and re-evaluated at a another time. This is a distinct advantage compared to ESI on-line coupling where the sample is permanently gone after the analysis.’

Despite the inherent incompatibility, several research groups have now come up with effective methods for coupling LC or CE to MALDI-MS. One such method was developed by Li and her colleagues, and utilizes a metal plate covered in tiny grooves. The idea is to fill the grooves with the MALDI matrix DHB and then slide the end of a capillary through the grooves, continually depositing analytes after separation by CE. This process mixes the analytes with DHB, allowing them to be analyzed by MALDI-MS.

Li used this coupling of CE and MALDI-MS to analyze a complex sample of neuropeptides, but found that CE on its own was not sufficient to separate all the neuropeptides, especially those with similar characteristics. So Li decided to add a LC stage before the CE stage, to produce the first ever coupling of multidimensional separation with MALDI-MS.


Crab neuropeptides

Testing this multidimensional system on another complex mixture of neuropeptides, derived from a crab, Li and her team first separated the neuropeptides by LC, splitting them into four different fractions. They then separated each of these fractions by CE, depositing the separated analytes in the grooves on the metal plate and then analyzing them by MALDI-MS.

In this way, they were able to detect 19 distinct neuropeptides in the mixture. In contrast, a combination of LC and MALDI-MS, in which the LC eluent is deposited as a series of spots prior to analysis by MALDI-MS, was only able to detect 14 neuropeptides. The five extra neuropeptides detected by the novel multidimensional system included two that had never been seen before.

Li attributes the better separation ability to a combination of the two separation techniques and the continuous collection of the separated analytes in the grooves on the metal plate, which allows the production of some useful visual information. ‘Because the system enables production of highly homogenous CE trace for MSI analysis with excellent reproducibility, the overlapped peaks from the complex samples can be easily analyzed as separated colored regions on the MS images,’ she told separationsNOW.

Although Li and her colleagues designed this system specifically for analysing neuropeptides, they are now considering applying it to other complex biological samples, such as cerebrospinal fluid and human serum. They are also looking at developing a fully-automated miniature device based on the system.

Related Links

Journal of Separation Science, 2012, 35, 1779–1784: "Neuropeptide analysis with liquid chromatography-capillary electrophoresis-mass spectrometric imaging"

Article by Jon Evans

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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