MALDI was developed for the ionisation of relatively large polypeptides and proteins but its application has widened to incorporate glycoproteins, oligonucleotides and complex carbohydrates. A great advantage of MALDI TOF MS is that the process of soft-ionisation causes little or no fragmentation of analytes, allowing the molecular ions of analytes to be identified, even within mixtures. Furthermore, if relatively pure material is available, unequivocal identification of that material can be achieved by a process known as mass-mapping. For example, mass-mapping of a protein is accomplished by breaking it into specific peptide fragments using amino acid specific proteolytic enzymes, the endoprotease trypsin being particularly useful. Mass spectrometric analysis of the enzymatic digest generates a 'mass-map' or profile which is unique to the analysed protein allowing unambiguous identification by the utilisation of database searches. In addition, structural characterisation can be realised by utilising options accompanying sophisticated instruments, e.g. post-source decay (see later).
MALDI TOF MS analysis is sensitive and very rapid as once the sample has been mixed with a 'matrix' on a MALDI target, a spectrum can be generated within seconds. Hence, the majority of protein and oligonucleotide analysis in the context of high throughput proteomics and genomics is carried out by MALDI TOF mass spectrometry.

The Instrument

The School MS Facility H&LS, KCL is equipped with a Bruker Daltonics Autoflex™ automated high-throughput MALDI TOF MS system. The instrument uses a 337 nm N2-laser to desorb and ionise molecules. The high resolution magnifying observation optics displays the target on the PC screen and enables exact positioning of the laser. Positive or negative ions are extracted into the 122-cm linear or 260 cm reflectron flight path using the delayed extraction mode. An accessory for post-source decay analysis is also available. In addition, the Autoflex™ uses targets in a microtitre plate format that may be interfaced to a robotic sample preparation line.

Instrument Mass Range and Sensitivity

MALDI TOF is particularly suitable for the analysis of high molecular weight compounds. including the determination of molecular masses of biopolymers such as peptides and proteins, carbohydrates, oligonucleotides and synthetic polymers with relative masses up to several hundred kilodalton. The upper mass limit though is not known, but it is probably not so much determined by the desorption/ionisation processes, but rather by ion detection efficiency. The lower mass limit of MALDI TOF MS is around 500 Da due to signals arising from molecular, fragment and adduct ions of the matrix. However, small molecular weight compounds (below 500 Da) can often be analysed without the addition of matrix in a technique called laser desorption/ionisation (LDI).
The term sensitivity in connection with MALDI is generally used to describe the smallest amount of analyte deposited on the MALDI target that can be detected. Sensitivity in this context does not refer to a concentration. However, as volumes between 0.2 and 1.5 uL are usually applied onto a MALDI target, the concentration range can be easily calculated, e.g. attomole MALDI sensitivity refers to picomolar concentration. Ions generated by MALDI can be routinely mass analysed in a TOF instrument in picomole (pmol = 10-12 mol) down to low femtomole (fmol = 10-15 mol) amount. However, attomole (amol = 10-18 mol) sensitivity has also been demonstrated using a MALDI TOF MS.

Applications of MALDI TOF MS

To date, MALDI TOF mass spectrometry has been successfully used for the analysis of a wide range of different analyte molecules. Important examples are peptides and proteins including glyco- and membrane proteins and phosphopeptides, oligosaccharides, oligonucleotides including SNP analysis, lipids, molecular aggregates with non-covalent interactions, complexes of metal ions with biomolecules, and synthetic polymers. MALDI TOF mass spectrometry is one of the most important tools for the analysis of the proteome, i.e. proteomics. In addition, it is the major tool for the analysis of the products of peptide synthesis.

Fundamentals of MALDI TOF MS

MALDI MS is a development of the direct laser desorption mass spectrometry of small organic molecules that was initially developed in the 1970s. MALDI MS was introduced in the late 80s when it was demonstrated that adding a small molecular weight organic matrix to an analyte could overcome molecular photodissociation of the sample ions induced by direct laser irradiation. The key aspect of MALDI MS is to dilute and isolate macromolecules in a suitable matrix of highly laser light absorbing small organic molecules, such as a-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA) and 2,5-dihydroxybenzoic acid (DHB), and then allowing it to dry on a MALDI-target into a crystalline deposit throughout which the molecules of the analyte are dispersed.

The excitation of the matrix by a high intensity laser pulse of short duration, the absorbed energy causing desorption (vaporisation) and ionisation of the analyte in a very dense MALDI plume. The ions are generated essentially at a point source in space and time then enter a vacuum where they are accelerated by a strong electric field in a 'flight tube' where they are then separated in time and finally hit the detector. An analyser measures the time-of-flight (TOF) taken for particular ions to hit the detector. The flight time of an ion is related to its mass-to-charge ratio (m/z), thus, mass spectra can be generated from simple time measurements. An example of a typical spectrum of a mixture of proteins is shown below.

MALDI TOF mass spectrum of a mixture of ubiquitin, cytochrome C and equine myoglobin using 2,5-dihydroxybenzoic acid (DHB) as the matrix. Also shown is the doubly charged species of cytochrome C.

Post-source Decay (a mode of operation on our School Instrument)

In principle, any fragmentation is undesirable if the analytical goal is to identify the molecular ion and to determine the mass of the biomolecule. However, MALDI ion fragmentation can be exploited to obtain structural information. Those ions, which fragment in the field-free region of the drift tube, retain essentially the same velocity as intact ions and cannot be distinguished from stable ions in a linear TOF instrument. This fragmentation is called post-source decay (PSD) and is a result of the laser irradiation and collision with other molecules (e.g. residual gas). The reflector will separate precursor and metastable decay ions by their difference in kinetic energy. PSD is routinely employed to obtain sequence information from peptides.

Delayed Extraction and Reflectron (choices on our School Instrument)

Delayed extraction (DE) is usually employed with our instrument and, as a result of the increased mass resolution, a better mass assignment can be achieved. Utilising DE (10 to several hundred nanoseconds) of ions can compensate for the initial velocity distribution of the MALDI generated ion packet such that same m/z ions arrive simultaneously at the detector. At the end of the 122 cm long linear flight tube of the Autoflex™ is located a reflector that compensates for the difference in flight times of the same m/z ions of slightly different kinetic energies (initial position and velocity dispersions). It consists of a series of evenly spaced electrodes onto which an electric field is applied. The spread in kinetic energy and velocity of the same m/z ions in the flight tube will result in different penetration depths into the reflector. Ions with greater kinetic energy arrive at the reflector first but penetrate deeper into the field thus travelling a longer flight path in the reflector than ions with less kinetic energy and arrive therefore at the detector at the same time. This correction leads to an increased mass resolution for all stable ions in the spectrum. For proteins beyond around 10,000 Da the peak broadening due to metastable decay is usually greater than the broadening due to kinetic energy distribution. For this reason, the use of the reflectron TOF is limited to the analysis of peptides and small proteins.

Dr Hendrik Neubert