MALDI – Matrix-assisted laser desorption/ionization

The analyte is mixed with a large excess of an aromatic ‘matrix compound’ that can absorb energy from the laser. The analyte and matrix are dissolved in an organic solvent and placed on a metallic probe or multiple-sample target. MALDI causes the ionization and transfer of a sample from the condensed phase to the gas phase via laser excitation of the sample. The solvent evaporates leaving matrix crystals in which the analyte is embedded.

Sample-matrix preparation procedures greatly influence the quality of MALDI mass spectra of peptides/proteins. Among the variety of reported preparation methods, the dried-droplet method is the most frequently used. MALDI is generally used with singly-charged ions with a high mass range analyzer such as TOF. MALDI can be used for high throughput microarrays on silicon chips, imaging of tissue or selection of individual cells or microorganisms.

MALDI requires samples to be dissolved in a matrix for analysis. Proteins are least soluble at their PI and therefore precipitate.

Detergents and salts should be reduced or removed from samples. Never use proteases and enzymes such as Cyanogen Bromide or trypsin in matrix preparation. They divide proteins.

Matrix crystal formation

Reference: http://www.chemistry.wustl.edu/~msf/damon/samp_prep_dried_droplet.html

Dried Droplet

In this method, the analyte and matrix solution are mixed together then loaded onto the MALDI sample plate. Solvents are dried by air drying. The disadvantage of this technique is the lack of signal reproducibility due to variability in drying conditions.

Fast Evaporation

Mix analyte with a specially prepared matrix. This produces small crystal with a very uniform surface, improving mass accuracy.

Overlayer

It involves a fast solvent evaporation step to form the first layer of small crystals, followed by deposition of a mixture and analyte solution on top of the crystal layer. This technique keeps the benefits of fast evaporation and reduces its disadvantages. Therefore it is more accurate and sensitive.

Matrix with nitrocellulose

When nitrocellulose is mixed with matrix solution, peptide ionization and signal reproducibility increases.

Ionization Mechanism

1. Matrix absorbs UV or IR energy from pulsed laser
2. Matrix ionizes and dissociates
3. Ions released by expanding plume

Matrix dependencies

• Different lasers work with different matrices
• Absorb energy from laser wavelength
• Most matrices are acidic by nature
• Matrix can be hot (likely to fragment peptides) or cold (not likely to fragment peptides)
• Require high matrix-analyte ratio
• Several crystallization methods exist

Hot or Cold

Cold --> DHB --> larger molecules and proteins --> creates fewer fragments
Hot --> CHCA (alpha-cyano) --> better with peptides --> creates more fragments

Delayed Extraction

Delayed extraction is a technique which allows ions to be extracted from ionization source after a cooling period of ~150 nanoseconds. This narrows the kinetic energy distribution of the ions, thus providing higher resolution than in continuous extraction techniques.

In delayed extraction mode, potential gradient does not exist when sample in ionized. Accelerating voltage is pulsed after a user-set time delay and ions are accelerated.

In continuous extraction mode, accelerating voltage is continuously applied, and the potential gradient exists when sample is ionized. Ions are accelerated immediately.

Delayed extraction improves mass resolution by reducing the spread in arrival times.

Usage

MALDI is widely used as a tool for peptides, proteins, and most other biomolecules (oligonucleotides, carbohydrates, natural products, and lipids). The utility of heterogeneous samples makes it very attractive for mass analysis of complex biological samples such as proteolytic digests. MALDI is predominantly used for the analysis of simple peptide mixtures, such as the peptides derived from a single spot from a 2D-gel. The utility of MALDI for biomolecule analysis lies in its ability to provide molecular weight information on intact molecules.

Soft ionization: not tearing the analyte apart. Soft ionization is capable of maintaining macromolcular complexes during ionization.

When MALDI does not work

Most charged or ionisable molecules interfere with the ionization of the analyte (i.e. compete for charges) and cause signal suppression and/or elevation of the background noise. These molecules include salts, chaotrophes, detergents, polymers, and all non volatile and ionic compounds.

Good crystallization is essential for good results. If salt or detergent impedes crystallization, you might get no signal.