subscribe
 

MALDI-TOF spectrometers have now become the instrument of choice

1st April 2013


Eric Russell looks at the increasing use of MALDI-TOFspectrometers in the laboratory environment and are nowthe instruments of choice for the analysis of proteins,peptides, oligonucleotides and synthetic polymers.

Performance of the MALDI-TOF technology was not as good as other analytical technologies when it first appeared in the 1950s and 60s, but continual development, combined with improvements in lasers and detectors, has today brought MALDI-TOF to the top.

Instruments are not cheap and prices depend on performance. Proteomics demands the highest level of performance although routine pharmaceutical analysis, for example, can be undertaken on spectrometers with a lower level of specification.

Mass spectrometers generally couple three devices: an ionisation device, a mass analyser, and a detector. Matrix Assisted Laser Desorption Ionisation (MALDI) is one of the most common ionisation techniques and is ideal for the analysis of large biomolecules.

Sample, or analyte, molecules are embedded in a matrix of small organic molecules that show a high resonant absorption at the laser wavelength used. The analyte is mixed with a matrix at molar ratios between 1000:1 to 10 000:1 of matrix to sample, to assist in the analysis of large, fragile molecules.

Matrix is a small organic compound such as sinapinic acid with certain properties such as high absorption of the laser beam that prevents the decomposition of fragile samples like proteins and oligonucleotides. The matrix molecules absorb the energy of the laser and gradually transfer it to the sample, preventing decomposition.

Matrix also promotes the ionisation process. Most matrices are acids and it is believed that ionisation is promoted by transferring a proton to the analyte.

Desorption

A critical step in the mass spectrometric (MS) characterisation of biological materials is the process of desorbing the analyte from a surface or matrix into the vacuum of the mass spectrometer. In the MALDI-TOF technique, a laser pulses into a sample of a light-absorbing matrix doped with analyte that results in a release of molecules into the vacuum of the mass spectrometer. The laser causes a small explosion that vaporises and ionises the sample.

In a time of flight (TOF) analyser, ions are separated by differences in their velocities as they move in a straight path toward a collector in order of increasing mass-to-charge ratio. Ions of different mass charge need different times to travel through a given distance in a field-free region after they have all been initially given the same translational energy.

A high electric field accelerates the ions down an evacuated tube in the mass spectrometer. As large particles travel more slowly than small ones, the mass of each ionised particle is determined by measuring its arrival time at the end of the tube. The system translates that speed into a very accurate molecular weight to enable identification.

In general, only molecular ions of the analyte molecules are produced and almost no fragmentation occurs. This makes the method well suited for molecular weight determinations and mixture analysis.

MALDI-TOF mass spectrometry separates and detects molecules by the intrinsic physical property of mass-to-charge ratio (m/z), which produces a molecular fingerprint. The coupling of solid-phase affinity methods with direct analysis by MALDI-TOF MS, an approach loosely referred to as affinity mass spectrometry, has greatly increased the speed and scope of the technology.

A spectrum of ion intensity as a function of the travel time is recorded by a special detector that notes the arrival time and intensity of signals as the groups of mass-resolved ions exit the flight-tube. Matrix ions and light peptides exit the flight tube first, followed by heavier proteins in increasing order of mass-to-charge ratio.

Technique of choice

MS is currently the only available methodology for the determination of accurate molecular weights of intact proteins, independent of size or post-translational modifications.

MALDI-TOF has rapidly emerged as the mass spectrometry technique of choice for the rapid screening of molecular weights of gel spots.

But knowledge of the pI and molecular weight of a protein spot rarely provides all the information required to identify a protein with a high confidence level. Other protein attributes are generally required and peptide mass fingerprinting has emerged as the fastest, simplest and overall most successful methodology for protein identification.

A mass spectrum of the peptide mixture resulting from the digestion of a protein by a proteolytic enzyme can provide a fingerprint that is specific enough to identify the protein uniquely. MALDI-TOF methodology has emerged as the best alternative for rapid screening of peptide maps.

It is relatively insensitive to contamination from buffer components such as salts and detergents.This allows peptide mixtures taken from in-gel or on-membrane digests to be analysed directly, with no pre-purification requirements.

There are a reasonably large number of well-characterised peptide and protein standards that are commercially available in pure form and that can be used for mass-axis calibration purposes. The highest degree of mass determination accuracy is achieved through internal calibration. The flight-times of the standard and unknown ions are measured from the same spectrum providing the best possible match of experimental conditions for the species involved.

MALDI-TOF technology is suitable for a wide range of applications from production through to research, and performance is related to cost. For use in proteomics, the highest level of performance and resolution is required. Most instrument manufacturers are developing products at this level from models that are currently appropriate for the routine analysis of large molecules in pharmaceutical and biotechnology development and clinical trials

Around the market

Applied Biosystems offers its Proteomics Solution 1. This is a comprehensive, automated system that accelerates the process of high throughput protein identification. Sample preparation, calibration and analysis are automated for unattended operation. The solution includes an online database and report generator.

The new Ultraflex TOF/TOF from Bruker Daltonic (Figs. 1 and 2) enables high-throughput protein identification by MALDI-TOF peptide mass fingerprinting, immediately followed by more detailed protein characterisation using MALDI-TOF/TOF tandem mass spectrometry (MS/MS) on the same prepared sample. Comprehensive MS/MS information is available from minute sample amounts within a few seconds.

Ultraflex TOF/TOF uses a patented gridless ion source and reflectron design, providing unsurpassed sensitivity in the standard MALDI-TOF MS mode.

With full microtitreplate compatibility and automated target supply by the Zymark Twister Microplate Handler for unattended target delivery, the Ultraflex TOF/TOF seamlessly interfaces with the biochemical lab and allows for unattended operation through day and night.

The system also incorporates the patented AnchorChip technology, which provides homogeneous, exactly-positioned samples on the MALDI target for robust and fast automation, as well as a sensitivity boost by an order of magnitude.

Ultraflex features a highly intuitive graphic user interface for simple operation on a PC while high magnification video optics provide excellent sample observation on screen. Data acquisition is automated and fast with no need to search for sweet spots.

The MALDI source is an N2 laser with 10-fold higher lifetime than standard lasers. Ultraflex also features extended self diagnostics with a remote service capability for on-line troubleshooting.

Routine analysis

GSG Analytical's future linear MALDI-TOF mass spectrometer is ideal for routine analysis and provides fast mass determination for large molecules of synthetic and pure polymers, proteins, carbohydrates, DNA and inorganic complexes.

The detector section includes a proprietary secondary ion generator for increased sensitivity at very high masses. An N2 laser is used and the quantities of sample needed for analysis are in the ultra-trace range. A robot preparation system is available. The software can be upgraded with time lag focussing and a high speed digitiser.

Kratos Analytical offers the Axima-CFR, a newly developed MALDI-TOF mass spectrometer ideal for a wide range of high throughput biochemical applications including proteomics and combinatorial chemistry.

The Kompact range includes two linear models: Alpha and Probe, with pulsed extraction for fast and accurate molecular weight determination for biochemical and biopolymer applications; and two linear/reflectron systems: Discovery and Seq, which are ideal for fast and precise molecular weight determination and structural analysis.

The company web site provides a series of useful hints and tips so users can get the most out of their instruments.

A new entrant into the market is the Mantis from Mass Technologies. The company says this is the first available desktop IR-MALDI mass spectrometer. An ultra-violet laser is also included in the instrument.

Mantis features a high precision linear x-y translational state for on-chip array sample analysis. The compact design also includes an optional sample cooling stage for utilising frozen matrices and end-cap reflectron and time lag focusing for better mass resolution.

Integrated solution

M@LDI is the primary MS data acquisition device of the Micromass ProteomeWorks System, an integrated protein function discovery solution. This has been developed in conjunction with Bio-Rad and represents a powerful solution for high throughput protein isolation and identification.

It is available in several popular formats; linear (L), reflectron (R), dual detector (LR) and high throughput (HT) - enabling the automated determination of intact protein masses and monoistotopic peptide mass fingerprints.

All M@LDI systems feature an automated laser positioning system for repeatable results. Micromass' proprietary MagLift robotic target plate loader allows batches of up to 50 plates to be analysed during a single run on a M@LDI HT instrument. This includes an integrated bar code reader to ensure correct identification of every target plate.

The Micromass TofSpec-2E (Figs. 3 and 4) is a compact high performance MALDI-TOF instrument. It is particularly suited to the characterisation of sub picomole quantities of biomolecules with molecular weights ranging from 500 Da to greater than 500 KDa. It may also be used to characterise high molecular weight synthetic polymer mixtures.

PerkinElmer is dedicated to developing sample-to-answer tools and innovative technologies that offer high productivity and performance. The OmniFLEX MALDI-TOF MS is a high-quality, benchtop system designed for the routine analysis of large molecules for a variety of applications in pharmaceutical and biotechnology development support, quality control and clinical trials.

Its high sensitivity and specificity makes the OmniFLEX an ideal choice of technology for the analysis of minute quantities of precious samples in the range femtomoles to picomoles. This includes proteins, peptides, oligonucleotides, oligosaccharides and synthetic polymers. OmniFLEX is the result of a partnership between Bruker Daltonics and PerkinElmer Instruments.

Compact automation

The SAI LaserToF range of MALDI-TOF mass analyser instruments is completely automated, compact and versatile, and designed to meet the needs of high throughput analysis.

Instruments have been designed for the rapid and accurate analysis of a wide range of biomolecules such as peptides, proteins and oligonucleotides.

The LT2 also has specialised chromatogram software for the analysis of thin layer chromatography (TLC) plates, which is of great use in applications such as impurity testing in the pharmaceutical industry.

Systems are complete with a fully automated sample introduction and analysis software with optimisation of sample position and laser power selected by the computer during the sample analysis.

A comprehensive toolkit for manipulating data is provided, with software modules to help interpret a variety of sample types from biomolecules to synthetic polymers.

The LaserTof LT3 is a bench top research grade MALDI-TOF instrument that includes a harmonic reflectron and a high mass detector as standard. It is compact, automated and versatile.

Price breakthrough

David Ames, sales and marketing manager for Stanford Research Systems, says the company has achieved a significant price breakthrough with its Profiler (Fig. 5) high-end MALDI-TOF spectrometer.

At under half the price of comparable models, Profiler offers a similar performance. Ames says it has created considerable interest at several exhibitions since its launch at the beginning of 2001. In conjunction with the booming proteomics market, Ames says the company is facing an excellent future.

Profiler is a bench top instrument withautomated operation. It features a 1.2m flight path and time lag focusing amongst a host of innovative features.

It is preloaded with instrument pre-settings optimised for every mass range. Users just have to choose the pre-settings and the only parameter that will need to be empirically adjusted is the laser intensity.

As proteomics moves from the life science of tomorrow to that of today, development of the enabling instrumentation is following and willensure that the full secrets of human life become ever closer.





Subscribe

Subscribe



Newsbrief

Twitter Icon © Setform Limited
subscribe