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.