The Importance of Controlled Concentration and Drying in MALDI-TOF Applications

Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is now an accepted and routine analysis technique for the elucidation and quantitation of biomolecules in life science research. In this technique, a coprecipitate of a UV-light absorbing matrix and a biomolecule are irradiated by a nanosecond laser pulse. The technique involves spotting small concentrated aliquots of material onto a matrix-coated target. The target is then positioned inside the mass spectrometer and the biomolecule of interest is desorbed from the matrix surface and ionized by the laser. Most of the laser energy is absorbed by the matrix, which prevents unwanted fragmentation of the biomolecule, while some of the energy causes ionization of the biomolecule. These ionized biomolecules are accelerated in an electric field and enter the flight tube of a time-of-flight mass spectrometer. During the flight in this tube, different molecules are separated according to their mass-to-charge ratio and reach the detector at different times. In this way, each molecule yields a distinct signal. The method is used for the detection and characterization of biomolecules such as proteins, peptides, oligosaccharides, and oligonucleotides, with molecular masses between 400 and 350,000 D. It is a very sensitive method, which allows the detection of low (10–15–10–18 mol) quantities of sample, with an accuracy of 0.1–0.01%. Although the technique can be very sensitive, concentrated samples achieve the best results.

Protein identification by this technique has the advantage of short measuring times (a few minutes) and negligible sample consumption (less than 1 pmol) together with additional information on microheterogeneity (e.g., glycosylation) and the presence of byproducts. Although molecular biology has provided powerful techniques for DNA analysis, this is not yet reflected in protein analysis. Genome sequencing has yielded a wealth of information on predicted gene products, but for the majority of the expressed proteins, no function is known. Proteomics is an important new field of study of protein properties, including expression levels, interactions, and post-translational modifications and thus can be described as functional genomics at the protein level. The mass accuracy of MALDI-TOF MS is sufficient to characterize proteins (after tryptic digestion) from completely sequenced genomes such as methanogens and yeast. The use of MALDI-TOF MS for the typing of single nucleotide polymorphisms using single nucleotide primer extension has also made important progress recently.

Oligonucleotides, proteins, antibodies, and other larger biomolecules are all suited to MALDI-TOF analysis. However, these compounds can be difficult to concentrate without exposing them to thermal damage or cross-contamination. MALDI spotters use nanoscale liquid handling to pipet drops of sample onto the precoated target, but the sample is picked up from a well in a fairly standard microplate. The bulk sample is frequently formatted in a 96-well plate, with a number of plates contributing to each MALDI analysis run.

Concentrating large biomolecules in such microplates is not straightforward, and it is here that the Genevac (Suffolk, U.K.) centrifugal evaporation technology can help. By protecting samples from overexposure to heat and by controlling cross-contamination in the plates, evaporators can significantly improve the results generated from MALDI-TOF analysis. This article looks at how that protection is achieved in practice.

Centrifugal evaporation is, of course, not new. The technique has been used in life science research for 20 years or more, but it was still rather crude until fairly recently. Samples were spun sufficiently fast (it was thought) to hold the sample in the bottom of the container as it boiled (evaporated), while atmospheric pressure was reduced to induce boiling close to (or below) room temperature. To speed up drying, heat could be applied by warming the chamber walls. More recently, manufacturers added powerful IR lamps to the system. These focus their IR energy onto the rotating sample, thus providing heat energy to the sample and speeding evaporation. The problems come with the behavior of complex biological mixtures in such a system, starting with the problems of overheating.

Figure 1 - A control sample of an oligonucleotide is analyzed by MALDI-TOF with spotting directly from the synthesis plate to the target.

Figure 2 - A second sample was spotted to the MALDI target, but in this case, the sample was preconcentrated on a Genevac HT-4. The results show good mass correlation, with no thermal damage to the oligo, due to the sophisticated sample temperature protection in the evaporator. (Data courtesy of Eurogentec S.A., Liege, Belgium.)

Heat energy is necessary to replace that lost as latent heat of evaporation in the boiling sample. As the solvent boils, it loses heat energy and cools itself and the container. This slows evaporation further, and thus energy must be directed into the drying sample to replace that which is lost, if a continuous evaporation rate is to be maintained. Infrared heater lamps, which are a development of halogen lamp technology, are very good at providing the necessary heat flow. However, they can sometimes be too efficient, leading to overheating of a sample that has already reached dryness. This is extremely undesirable where proteins and peptides are concerned, since they are thermally labile and easily damaged by temperatures above 40 °C. In order to prevent this situation, it is necessary to be able to measure the temperature of the sample as it spins around. Although that allows control of the heat energy flowing in, it is difficult to accomplish. Many manufacturers gave up at this point and chose to control only the temperature of the chamber wall itself, but this is extremely unsatisfactory and provides no direct information on the physical status of the sample (Figures 1 and 2).

This problem was overcome in the EZ-2 concentrator/dryer (Genevac) by using a finely tuned IR pyrometer combined with sturdy, solid aluminum sample holders. The noncontact sensor measures the surface temperature of the aluminum as it passes by and can be accurate to ±2.5 °C, which is adequate for this application. Because heat flow through the aluminum sample block is uniform, and because the instrument can control the heat flow to the samples by switching the IR lamps on or off, it is then possible to deduce the actual sample temperature from these data using a simple algorithm. In this way, the EZ-2 allows scientists to preselect a sample protection temperature suitable for biology applications—normally 35 or 40 °C.

The second problem for highly sensitive samples such as DNA, protein isolates, or peptides is one of contamination. While great care may be taken at the spot-picking, excision, and loading stages to avoid cross-contamination, the sample microplates present a unique problem at the concentration stage. In a conventional evaporator, the plates may only be spinning at 250–300 ×g. Independent trials by GlaxoSmithKline (Harlow, Essex, U.K.) have shown that this level of g-force is insufficient to entirely prevent cross-contamination within the plate. Contamination arises as samples begin to bump during the evaporation process. Bumping is a widely misunderstood phenomenon that is the major cause of spoiled or contaminated samples in such applications. It can be eliminated by the use of the DriPure™ bumping control system (Genevac). With DriPure enabled, the vacuum is gently ramped down over a period of 30 min or so, while at the same time, the applied g-force is increased to well over 450 g in order to prevent bumping from occurring, by accentuating the boiling point/depth gradient and concentrating all the “hot enough to boil” solvent near the surface. This also creates active convection and ensures good mixing so that temperature gradients do not arise that could cause chaotic mixing of areas of liquid of dissimilar temperature. DriPure also ensures that any material that may eventually be ejected from the liquid surface is kept within the plate well.

GSK studies showed that with DriPure activated, bumping was eliminated, even for difficult solvent/solute mixtures in multiple-well plates, such as acetonitrile/water HPLC fractions and dichloromethane (DCM)/methanol mixtures.

Another advantage of using the EZ-2 when preconcentrating samples in this way is the ability to achieve higher spotting densities, leading to greater sensitivity for low-expression proteins, without complicated liquid handling procedures involving repeatable nanospotting.

Combining these obvious benefits with ease of use has made the EZ-2 popular with researchers around the world. Scott Dixon, Senior Researcher at UCSF Cancer Research Institute (San Francisco, CA) has had an EZ-2 working within his peptide laboratory and specifically with the MALDI-TOF facility for some time now. According to him, it is very easy to use. Users spot when they perform MALDI applications as well as electrospray applications. Because concentration is important in the spots, they use the EZ-2 concentrator to get the amount of material spotted to be consistent. ICAT labeling allows them to obtain quantitative information from the mass spectrometer. It requires them to dry down peptides completely, and in this respect, the EZ-2 is very useful. It has helped improve results, and by speedily concentrating or drying a number of plates simultaneously, helps them better utilize the MALDI. The EZ-2 performs the function that was previously done by lyophilization. Lyophilization was very slow; hence, using the EZ-2 to dry down spots is much quicker.

Mr. Knight is Marketing Manager, Genevac Ltd., Farthing Rd., Ipswich IP1 5AP, U.K.; tel.: +44 1473 243030; fax: +44 1473 742987; e-mail: [email protected]. U.S. contact: Mike Ward, Genevac USA, 707 Executive Blvd., Ste. D, Valley Cottage, NY 10989, U.S.A.