MALDI Technique

Biomolecular mass spectrometric techniques (MALDI- and ESI-MS) for the analysis of biopolymers are leading representatives of biochemical techniques. The sensitivity of biomolecular... 

To benefit general readers, the discussion has been limited to methodologies that are accessible to nonspecialists and that can be carried out on commercially available spectrometers without special modifications. The chapter illustrates the principles of mass spectrometry by demonstrating how various techniques [MALDI, ESI, Fourier transform ion cyclotron resonance (FT-ICR), ion traps, and tandem mass spectrometry (MS-MS)] work. It also provides examples of utilizing mass spectrometry to solve biological and biochemical problems in the field of protein analysis, protein folding, and noncovalent interactions of protein-DNA complexes. 

The liquid volume of a sample required for analysis depends on the ionization technique, MALDI or ESI, and the introduction technique (see Table 4.1). The following statements assume that we are analyzing a sample near the detection limit of the analyte in a specific mass spectrometer. For MALDI-MS, the researcher typically spots 0.1 to 1 jL onto the MALDI sample plate. Thus, a minimum starting volume of 1 of 5 jL of sample is recommended. For ESI, the required sample volume is primarily dependent on the sample introduction technique. If the researcher uses a nanoflow electrospray technique, capillary EC, or capillary electrophoresis, then typically a l-pL voliune is required. However, larger sample volumes are recommended for ease of handhng. If the voliune is small, then the analysis may be limited to one experiment when additional MS or MS-MS experiments are desired. For higher flow rate ESI sources, the researcher should supply 50 pL or more for direct infusion experiments or for loading 5 to 20 pL onto an analytical EC column. 

High-resolution mass spectrometiy methods have recently been used to discriminate bacterial isolates through the characterisation of specific cellular components. One of these techniques, MALDI-TOF MS, has been... 

Among the mass spectrometric techniques, MALDI-MS is the least susceptible to the presence of salts and detergents in the sample. However, high levels of contaminant may lead to poor crystalization of the matrix and consequently, a... 

Among the various mass spectrometry techniques, MALDI is probably the most important as it provides an absolute method for molar mass determination and molar mass distribution, as well as information on end groups and copolymer composition. The MALDI process consists of the ablation of the polymer molecules dispersed in a matrix typically made up of aromatic organic acids. The matrix needs to be able to absorb at the wavelength of a laser (usually 337 nm). This process excites the matrix molecules, which vaporize at the same time, the polymer molecules desorb into the gas phase, where they are ionized. Thus, the role of the matrix is that of transferring the laser energy to the polymer molecnles. 

In the mass spectrometric technique, MALDI (matrix assisted laser desorption/ionization), the polymer sample is dissolved, the solutions atomized so that a single polymer chain is contained in a droplet. The droplets are then ionized and pumped into a mass spectrometer where they are accelerated and separated based on their mass. The resulting spectrum is based directly on the molecular weight of the polymer chain... 

Of the fragmentation techniques MALDI-ISD, ECD, and ETD, it is only the latter that is directly compatible with the requirements and timescale of an online HX-MS workflow. By combining the classic bottom-up HX-MS workflow with gas-phase fragmentation by ETD, detailed information on protein HX can be obtained [47,57]. In such a combined workflow, enzymatic solution-phase cleavage is followed by automated (data-dependent acquisition) or manual (targeted) selection of peptides for gas-phase cleavage by ETD [57] (Eigure 8.6). 

Technique MALDI Matrix-Assisted Laser Desorption... 

The collection of data and critical evaluation of possible influences of parameters on the CLD (as was carried out by Heuts et al. ) might help to elucidate the current question of the true nature of chain length dependence of the rate constant of propagation in free radical polymerization. Therefore, the investigation of the polymerization behaviour of monomers other than styrene and methyl methacrylate is necessaryf and the use of the correction procedures should eliminate the error introduced by the effect of BB. Thus, comparison of data obtained from either different research groups and/or with the aid of different techniques (MALDI, SEC) should be better feasible. 

At high laser powers only atomic (elemental) information is obtained, however, at lower power densities, close to threshold, molecular information can be obtained, although extensive fragmentation is frequently observed. By careful control of the initial conditions and laser energy this approach has been refined to allow even delicate biological molecules, such as proteins, to be ejected from a solid or liquid into the gas phase and ionized, without fragmentation. Indeed, this is the basis of the now well-established technique MALDI, which is described in further detail below. 

In order to circnmvent this limitation but still preserve the detailed molecular weight information generated by this powerful detection technique, MALDI-TOF has been nsed as an absolute detector, both on-line and off-line, for... 

In this article, focus is on the soft ionization technique MALDI, which is mainly in use for synthetic polymers. ESI is briefly discussed, although the electrospray method has been generally conflned to polar polymers, which are water-soluble. Other ionization methods that have been used on synthetic polymers, mainly laser desorption (LD) and fleld desorption (FD), are confined to low... 

Catania (Italy) Author of more than 50 publications and of 18 international invited lectures. He is currently working in the field of characterization of polymers and copolymers. He is an editorial board member of Rapid Communications in Mass Spectrometry. Research interests Structural characterization of polymers by mass-spectrometric techniques MALDI for the analysis of polymers and copolymers chain statistics applied to copolymer sequence analysis MonteCarlo simulations Bivariate distributions of chain size, and composition in high conversion copolymers. 

The peptide at m/z 1717.15 is moreover a very good example to illustrate the fact that both techniques (MALDI and ESI) and instruments (QIT/RTOF and QIT) complement each other to corroborate and supplement the results (Fig. 6). After interpretation of the MALDI CID spectra sequence, uncertainties due to too many possible dipeptide masses in the low mass range existed. However, missing information on parts of the sequence elucidated from the nano-ESI CID spectrum could be unequivocally reassigned after taking the information from the MALDI MS/MS spectra into account. 

In most published techniques MALDI mass spectroscopy and SEC are not directly coupled off line. For further discussion see Chapter 5. An exception to this is the work of Esser and co-workers [71] in which the two units are interfaced via a robotic interface. This technique was applied to PS, PMMA and butyl (methacrylate-methyl methacrylate) copolymer. Mehl and co-workers [88] combined SEC with MALDI-MS to provide accurate molecular weight determinations on polyether and PU soft blocks. 

MS is also used for the tinalysis of the more abundant (non trace) components of food, e.g. oils and fats (triglycerides), proteins and carbohydrates. Analysis of these materials is often challenging, as they may comprise complex mixtures of isomeric compounds. Modern MS techniques (MALDI TOP and electrospray ionization) have become extremely important in protein and peptide studies. 

Both ionization techniques, MALDI and ESI, deliver best results if the samples are pure and concentrated. In both cases, peptide and protein samples usually enter the sample preparation as aqueous solutions, which can contain varying amounts of an organic solvent, most often acetonitrile, methanol, or ethanol, and are usually acidified (pH 1-3). Acidification is, with only a few exceptions, a prerequisite for MALDI but not for ESI, which is far more flexible regarding the pH of the sample solution if appropriate (compatible) buffer systems and ion-pairing agents are used. 

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