Sample Preparation Methods for MALD1 Experiments

Successful analysis of compounds by matrix-assisted laser desorption/ionisation depends on a number of different factors In particular, the choice of matrix material and sample preparation method are o f great importance. The matrix must have a strong absorption coefficient in the wavelength of the incident laser beam and, in most cases, this is in the ultra-violet region of the spectrum at ~ 337nm. A homogeneous sample preparation aids the efficiency of the MALDI process, so the ability o f the analyte to mix effectively with a suitable matrix material in solution is also important. Matrix materials also have to fulfill the criteria of being stable under vacuum conditions Many of the matrices used for the analysis of biomolecules are aromatic acids which have labile protons, and initial criteria for the choice o f a suitable matrix material included the need for acidic protons Growth in this area of research has been rapid and many materials which do not contain such a function have been reported to act succesfully as matrices. Materials which are either liquid or solid under standard temperature and pressure have been used as matrices

Biomolecules such as peptides, carbohydrates and oligosaccharides readily form |M+H]+ ions by the MALDI process, however, synthetic polymers are usually detected as cation-attached oligomer ions in the form |M+X] +, where X is usually an

alkali metal in the case of such polymers as poly(ethylene glycol) and poly(methyl methacrylate) or a transition metal cation in the case of poly(styrene).

General sample preparation procedures for the analysis o f both polymers and biomolecules are outlined in this section.

2.2.1.1 Traditional MALDI Sample Preparation

i) lliom olecules

Compounds such as insulin and bradykynin were used as calibration materials. A solution of the analyte (-lO '^M in acetone or acetonitrile / water solution) was mixed 1:1 with suitable matrix, usually sinapinic acid or alpha-cyano hydroxy cinnamic acid. A 1 pi spot o f the sample mixture was spotted onto the stainless steel sample slide and dried off in a stream o f warm air.

ii) Polymers

A solution o f polymer (~10"^M, 3-5 mg per ml in THF) was doped with a solution of cation salt (0 .1M, l-100pl in THF) Equal quantities if this mixture and the matrix solution (0 .1M in THF) were mixed and lpl o f this mixture was deposited onto a stainless steel sample slide and the solvent was evaporated off under a stream o f warm air.

There are many other techniques which can be used to affect the results obtained by MALDI. For instance, air drying the solution gives different results form stirring during air drying7*5 Other methods such as two-layered deposition7®5’ spin-coated drying7*7 or crushing the crystals7** 7*51 after drying have been proposed to improve sample homogeneity. Electrospray (ES) as a sample prepration method was also investigated as part o f this re se a rc h90

2.1.1.2 Electrospray Sample Preparation

The electrospray set-up consisted of a 100f.il sample-container feeding the analyte solution through a needle placed at at high potential relative to an adjustable grounded sample-table The potential applied to the needle was - 8kV and the distance from the needle to the sample table was between 2 and 4 cm. A wire which was adjusted upwards and downwards was inserted into the needle to regulate the flow through the needle. The flow rate of the analyte solution was determined by measuring the time taken to empty the lOOpI container A positive electric field was used to drag the solution out from the needle into a cone, the apex o f which was enhanced with positive charge. An equal flow of solution was emitted from the apex of the cone in the form o f micrometer-sized positively-charged species.

The experimental procedure was concerned with optimising both the distance between the needle and the sample table and the needle potential , such that a single spray was emitted from the apex of the liquid cone. These sprayed droplets had to evaporate before they hit the MALDI sample slide located on the sample table

A single spray ensured spot-to-spot reproducibility, however, the needle potential could be increased so that multi-sprays were emitted from the vertex of the cone and this function was used to decrease spraying time. Single sprays produced a circular spot which was — 1 cm in diameter whereas multi-sprays covered an area of 3cm in diameter with many domains Work carried out with this apparatus used only the single-spray function.

A strong light positioned between the operator and the electrospray apparatus was used to observe the spray-cone to optimise the conditions. The needle potential was gradually increased until a single-cone was observed, and, if the sample slide was viewed at an angle, it was possible to see the formation of a thin layer within a few seconds A schematic diagram of the electrospray apparatus is shown in Figure 2.10

The conditions under which the samples were electrosprayed were carefully monitored. A typical polymer sample spot would take between 2 and 3 minutes to spray at a needle potential o f - 6kV.