MALDI-TOF MS: Much More than only Protein Analysis!

Carbohydrates and particularly lipids represent molecules with significant structural variabilities: this is stemming from (a) the headgroups, (b) the different fatty acyl residues and (c) the linkage types (acyl-acyl-, alkyl-acyl-, and alkenyl-acyl-). Therefore, analytical methods to clarify these aspects are of immense significance. As the amount of the available biological material is normally limited, suitable methods should also exhibit reasonable sensitivities. It is our aim to introduce MALDI (matrix-assisted laser desorption and ionization) MS (mass spectrometry) [1] (often but not necessarily with a time-offlight (TOF) mass analyzer) as a simple and sensitive analytical method that helps to overcome many problems related to carbohydrate and particularly lipid analysis [2]. Although the history of MS dates back to the end of the nineteenth century, applications of MS to the structural analysis of biomolecules are rather new: the MS analysis of such complex molecules (often in combination with high polarity and low volatility) became only possible with the invention of “soft ionization” methods such as electrospray ionization (ESI) and “MALDI” [3]. Both techniques are nowadays widely used, whereby the focus is often on the investigation of proteins - normally subsequent to enzymatic digestion of the (unknown) protein into characteristic peptides, the MS of which enables the elucidation of the protein sequence and further structural properties [4]. It is a characteristic property of “soft ionization” MS that there is only a small extent of analyte fragmentation and, thus, the intact ions of the analyte of interest can be observed. This is a pronounced difference in comparison to the classical “electron ionization” (EI) technique. Although MALDI MS has many advantages it should be noted that the analysis of small molecules (for instance, free fatty acids) is still a problem - despite many improvements that could be recently achieved [5]. This problem with small molecules can be easily understood when the principle of MALDI MS is considered: MALDI MS is based on laser ionization, whereby normally UV lasers (emitting often at λ=337 nm) are used. Since not all potential analytes exhibit a sufficient absorption at this wavelength, a “matrix” must be used [1]. This matrix is normally a small organic molecule (such as 2,5-dihydroxybenzoic acid (DHB)) [6] that absorbs the laser energy, is evaporated and simultaneously carries the analyte into the gas phase (high vacuum). In the gas phase collisions between the analyte and cations (such as H + or Na + ), that are either already present in the lipid extract of interest or artificially added, occur leading to ion generation. As the mass of the observed (positive) ions is slightly higher than the mass of the analyte, these are normally termed “adducts” or “quasimolecular” ions. Of course, analytes with acidic groups are also detectable as negative ions. Since the matrix is normally in considerable excess over the analyte (at least 100:1), it is not surprising that the smaller mass range is dominated by matrix ions [7]. This is the reason why the small mass range is normally not shown when MALDI spectra are discussed. Despite this drawback, however, there are a lot of advantages: 1. The necessary sample preparation is simple and fast - and comprises often only the mixing of the analyte with the matrix and the deposition of this mixture onto the sample carrier, the “target”.