The MALDI Process and Method
Franz Hillenkamp, Thorsten W. Jaskolla, and Michael Karas
Matrix-assisted laser desorption/ionization ( MALDI ) is one of the two "soft" ionization techniques besides electrospray ionization ( ESI ) which allow for the sensitive detection of large, nonvolatile and labile molecules by mass spectrometry. Over the past 27 years, MALDI has developed into an indispensable tool in analytical chemistry, and in analytical biochemistry in particular. In this chapter, the reader will be introduced to the technology as it stands now, and some of the underlying physical and chemical mechanisms as far as they have been investigated and clarified to date will be discussed.
Attention will also be focused on the central issues of MALDI, that are necessary for the user to understand for the efficient application of this technique. As an in-depth discussion of these topics is beyond the scope of this chapter, the reader is referred to recent reviews [1-4]. Details of the current state of instrumentation, including lasers and their coupling to mass spectrometers, will be presented in Chapter 2 .
As with most new technologies, MALDI came as rather a surprise even to the experts in the field on the one hand, but also evolved from a diversity of prior art and knowledge on the other hand. The original notion had been that (bio)molecules with masses in excess of about 500-1000 Da could not be isolated out of their natural (e.g., aqueous) environment, and even less be charged for an analysis in the vacuum of a mass spectrometer without excessive and unspecific fragmentation. During the late 1960s, however, Beckey introduced field desorption ( FD ), the first technique to open a small road into the territory of mass spectrometry ( MS ) of bioorganic molecules . Next came secondary ion mass spectrometry ( SIMS ), and in particular static SIMS, as introduced by A. Benninghoven in 1975 . This development was taken a step further by M. Barber in 1981, with the bombardment of organic compounds dissolved in glycerol with high-energy atoms, which Barber coined fast atom bombardment ( FAB ). It was in this context, and in conjunction with the first attempts to desorb organic molecules with laser irradiation, that the concept of a "matrix" as a means of facilitating desorption and enhancing ion yield was born . The principle of desorption by the bombardment of organic samples with the fission products of the 252Cf nuclear decay, later termed plasma desorption ( PD ), was first described by R. Macfarlane in 1974 . Subsequently, the groups of Sundqvist and Roepstoff greatly improved the analytical potential of this technique by the addition of nitrocellulose, which not only cleaned up the sample but was also suspected of functioning as a signal-enhancing matrix .
The first attempts at using laser radiation to generate ions for a mass spectrometric analysis were reported only a few years after the invention of the laser [10, 11]. Vastola and Pirone had already demonstrated the possibility of recording the spectra of organic compounds with a time-of-flight ( TOF ) mass spectrometer. Subsequently, several groups continued to pursue this line of research, mainly R. Cotter at Johns Hopkins University in the USA and P. Kistemaker at the FOM Institute in Amsterdam, the Netherlands. Indeed, for a number of years the Amsterdam group held the high-mass record for a bioorganic analyte with a spectrum of underivatized digitonin at mass 1251 Da ([M + Na]+), desorbed with a CO2-laser at a wavelength of 10.6 mim in the far infrared ( IR ) .
Independently of, and paral