MALDI laser pulse

Matrix-assisted laser desorption mass spectrometry (MALDI-MS) is, after electrospray ionization (ESI), the second most commonly used method for ionization of biomolecules in mass spectrometry. Samples are mixed with a UV-absorbing matrix substance and are air-dried on a metal target. Ionization and desorption of intact molecular ions are performed using a UV laser pulse. 

A lot of features of MALDI are conveyed by its name it is a desorption ionization, produced by a laser beam, and assisted by a matrix (Figure 2.5). The analyte (1 pmol or less) is mixed with a suitable matrix in a 1 1000 or higher molar ratio. The matrix is composed of a compound with a strong absorption at the wavelength of the laser used. These two factors, matrix excess and its strong absorption, ensure that the energy from the laser pulse is absorbed by the matrix and not by the analyte, thus avoiding its decomposition. Nicotinic acid, sinapinic acid (SA), 2,5-dihydroxy benzoic acid (2,5-DHB) and 2-(4-hydroxyphenylazo)benzoic acid (HABA) are some of the most commonly used matrices for MALDI. 

Note The fluence is defined as energy per unit area in MALDI typical flu-ences are in the range of 10-100 mJ cnT. The irradiance is fluence divided by the laser pulse duration in MALDI the irradiances are in the range of 10 -... 

Menzel, C. Dreisewerd, K. Berkenkamp, S. Hillenkamp, F. The Role of the Laser Pulse Duration in Infrared MALDI-MS. J. 

After introduction of the target into the vacuum, an UV laser pulse is used to desorb and ionize the sample. Nitrogen laser emitting at 337 nm and Nd YAG laser emitting at 355 nm are the most widely used. MALDI is a very powerful technique for the analysis of synthetics and natural biopolymers. It has completely replaced former techniques such as fast atom bombardment (FAB). In... 

As the laser pulse is in the nanosecond range, a fast mass spectrometer has to be coupled in series. In most cases, MALDI is coimected to a time-of-flight (TOF) mass spectrometer with which m/z ratios are determined by precisely measuring the time an ion needs to pass from the ion source to the detector. Besides its abil-... 

Fig. 8.2 Principle of the MALDI process. Initially, analyte and matrix are co-crystal I ized. After evaporation of the solvent, a nanosecond laser pulse is directed onto the crystalline surface, and both matrix and analyte molecules are desorbed. A complex reaction cascade leads to the formation of charged analyte molecules that reach the mass spectrometer without significant fragmentation.

I 8 Laser Desorption Assays - MALDI-MS, DIOS-MS, and SAMDI-MS taser pulse laser pulse... 

In a time-of-flight (TOF) analyzer the time of flight of ions between the ion source and the detector is measured [61]. This requires that the time at which the ions leave the ion source is well-defined. Therefore, ions are either formed by a pulsed ionization method or various kinds of rapid electric field switching. The single discontinuous laser pulses at distinct time points used in MALDI can be ideally combined with time-of-flight mass separation. TOF analyzers thus received increasing interest with the development of MALDI MS. The schematic draw of a linear MALDI-TOF MS is shown in Fig. (9). 

A schematic of the basic principles of a matrix-assisted laser desorption/ion source is shown in Figure 2.35. By the interaction of a focused laser beam with short pulses and a suitable matrix, the energy of the photons is transferred to the matrix molecules. In MALDI mostly pulsed UV (e.g., nitrogen, X = 337 nm, pulse duration 3-10 ns), but also IR lasers (e.g., Er YAG, X = 2.94 (xm or C02, X = 10.6(xm with a higher pulse duration of up to 600 ns) are used. The MALDI mass spectra obtained during soft ionization by UV and IR lasers are identical. The energy density... 

This desorption ionisation technique leads to weak fragmentation. The analyte is incorporated into a solid organic matrix (such as hydroxybenzoic acid) and the mixture is placed on a sample holder that is irradiated with UV laser pulses (e.g. N2 laser, A = 337 nm, pulse width = 5 ns). The laser energy is absorbed by the matrix and transferred to the analyte, which becomes desorbed and ionised (Fig. 16.18c). Although MALDI is considered to be a soft ionisation technique, a substantial amount of energy is involved. Because the technique involves pulsed ionisation, it is well suited for time-of-flight mass analysis of biomolecules. The analysis of small molecules (M < 500 Da) is limited because the matrix decomposes upon absorption of the laser radiation. However, solid supports such as silicone can be used as the matrix to overcome this disadvantage. 

MALDI is considered a surface analysis technique and a relatively nondestructive ionization technique (Page and Sweedler, 2002). Typically, a sample spot on the target can be assayed multiple times because only a small fraction is vaporized for each laser pulse. However, no further spectra are detected after a completed ablation by the laser in MALDI. The depleted amounts were observed to be associated with the sample identity, sample spot size, and MALDI matrices (Page and Sweedler, 2002). 

In 2000, various developments in the field of MALDI led to the advent of new methods such as the atmospheric pressure MALDI (AP-MALDI) source. This method combines the atmospheric pressure (AP) source and MALDI [45-47]. Indeed, this source produces ions of analytes under normal atmospheric pressure conditions from analyte-doped matrix microcrystals by irradiating these crystals with laser pulses. 

Principle of an LTOF instrument tuned to analyse positive ions produced by MALDI. After their formation during a laser pulse, ions are subject to the applied electric field. Ions are continuously accelerated and drift in a free-field region. They travel through this region with a velocity that depends on their m/z ratios. Ions are thus dispersed in time. 

If delayed extraction increases the mass resolution without degradation of sensitivity compared with continuous extraction, it also has limitations. Indeed, delayed extraction complicates the mass calibration procedure. It can only be optimized for part of the mass range at a time and is less effective at high mass. Delayed extraction partially decouples ion production from the flight time analysis, thus improving the pulsed beam definition. However, calibration, resolution and mass accuracy are still affected by conditions in the source. For instance, in the usual axial MALDI-TOF experiments, optimum focusing conditions depend on laser pulse width and fluence, the type of sample matrix, the sample preparation method, and even the location of the laser spot on the sample. 

The speed of MALDI analysis depends on the laser pulse rate. With the recent introduction of 200-Hz lasers, samples can be analyzed ten times faster than before. This development is especially advantageous for offline liquid chromatography (LC)/MALDI applications (Ericson et al., 2003). MALDI mass analysis is performed considerably faster than LC separation, allowing for chromatographic... 

A recently introduced technique for the separation of larger molecules is matrix-assisted faser Resorption-ionisation mass spectrometry (MALDI-MS). Developed by Karas et al. [4, 5] in 1988, it has been successfully used to determine the mass of biomolecules up to 500.000 Da. This method is based on the principle that the dissolved specimen is mixed with a matrix, and then crystallizes. After drying, a laser pulse is directed onto the solid matrix to photo-excite the matrix material,resulting in desorption and soft ionisation of the analyte.The molar mass is then determined by the lime ef ilight (TOF). 

Currently developed for many applications (Stoeckli et al. 1999 Stoeckli et al. 2001, 2002,2003 Chaurand et al. 2004), MALDI MSI is achieved by rastering sequentially the surface of a defined area while acquiring a mass spectrum from every location (see Figure 2). Atypical sample preparation for MSI involves the fixation of the sample, for example, tissue section, on a MALDI plate and the application of the matrix solution over the latter, either as a thin layer or as a spot pattern, to get co-crystallization of analytes with matrix while solvents evaporate. Once dried, the sample is introduced in the mass spectrometer, where, for each defined image position, short UV laser pulses are fired onto the surface to generate ions. Those are analyzed by the TOF instrument and a mass spectmm is acquired. 

Figure 4.16. MALDI-TOF Mass Spectrometry. (1) The protein sample, embedded in an appropriate matrix, is ionized by the application of a laser beam. (2) An electrical field accelerates the ions formed through the flight tube toward the detector. (3) The lightest ions arrive first. (4) The ionizing laser pulse also triggers a clock that measures the time of flight (TOF) for the ions. [After J. T. Watson, Introduction to Mass Spectrometry, 3d ed. (Lippincott-Raven, 1997), p. 279.]...

MALDI analysis was conducted on an aliquot of the original digest mixture. Mass measurements were made with a Bruker MALDI/TOF MS instrument (Billerica, MA) equipped with a nitrogen laser (337 nm). Spectra were averaged from 100 to 150 laser pulse samples, a-cyano-4-hydroxycinnamic acid was used as the MALDI matrix. Samples were prepared from the CytC digest solution, and acetonitrile and water (50 50) which was 0.1% in TFA. 

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