MALDI contaminant

In direct insertion techniques, reproducibility is the main obstacle in developing a reliable analytical technique. One of the many variables to take into account is sample shape. A compact sample with minimal surface area is ideal [64]. Direct mass-spectrometric characterisation in the direct insertion probe is not very quantitative, and, even under optimised conditions, mass discrimination in the analysis of polydisperse polymers and specific oligomer discrimination may occur. For nonvolatile additives that do not evaporate up to 350 °C, direct quantitative analysis by thermal desorption is not possible (e.g. Hostanox 03, MW 794). Good quantitation is also prevented by contamination of the ion source by pyrolysis products of the polymeric matrix. For polymer-based calibration standards, the homogeneity of the samples is of great importance. Hyphenated techniques such as LC-ESI-ToFMS and LC-MALDI-ToFMS have been developed for polymer analyses in which the reliable quantitative features of LC are combined with the identification power and structure analysis of MS. 

The type of proteinaceous binder was correctly identified in all model samples. In only one case (S10), the animal glue was additionally identified, although the restorer who prepared these model samples declared that the sample contained only egg binder. It is possible that this sample was contaminated during its preparation or during laboratory treatment. The results indicate that this method does not allow reliable identification of the presence of individual egg yolk and egg white most probably it is caused by the presence of a trace of egg white that is always present in the egg yolk preparations (and vice versa) and can be detected by the highly sensitive PMM method. The identification of individual types of animal glues will never be reliable by MALDI-TOF mass spectrometry because of their similar composition the application of ESI (electrospray ionisation)-MS/MS (Section 6.5) could possibly overcome this problem. Only the fish glue, whose peptide... 

The easiest way to detect a protein modification seems to be the mass measurement of all peptides generated by enzymatic digestion. The comparison with the predicted peptide masses from the sequence of the protein identifies unmodified peptides and unexplained masses would give indications to modified peptides. Unfortunately, this is not a suitable approach in practice. In many peptide mapping experiments done with the MALDI mass mapping technique, up to 30% of the measured masses remain unexplained. This is probably due to protein contaminations from human keratins, chemical modifications introduced by gel electrophoresis and the digestion procedure, and other proteins present at low levels in the piece excised from the sodium dodecyl sulfate gel. The detection of a protein modification requires a more specific analysis. 

If an analyte is definitely insoluble or only soluble in solvents that are not acceptable for the standard MALDI sample preparation technique, it can alternatively be ground together with the solid matrix, preferably in a vibrating ball mill. The resulting fine powder is then spread onto the target. To avoid contamination, nonadherent material should be gently blown away from the target before insertion into the ion source. [103,108,109]... 

The quality of the sample influences the quality of the data obtained by MALDI-TOFF. Contaminations inhibit ionization. As no in-line HPLC system is included in the system, contaminants cannot be removed from the sample. Fortunately, some new clean up tools (e.g., ZipTips) for removal of contaminants have been developed recently (Figure 5.5). 

With respect to sample preparation, it is necessary to develop effective and fast procedures involving only a few steps in order to avoid contamination, reduce analysis time and to improve the quality of analytical work. Microsampling and the use of smaller sample sizes is required and also the further development of analytical techniques. In particular, there is a need for the development of online and/or hyphenated techniques in ICP-MS. Microsampling combined with the separation of small amounts of analytes will be relevant for several chromatographic techniques (such as the development of micro- and nano-HPLC). There is a demand for further development of the combination of LA-ICP-MS as an element analytical technique with a biomolecular mass spectrometric technique such as MALDI- or ESI-MS for molecular identification and quantification of protein phosphorylation as well as of metal concentrations, this also enables the study of post-translational modifications of proteins, e.g. phosphorylation. 

Methanol is a common solvent for extracting fiavonol glycosides from plant material. This procedure involves a simple, rapid, and efficient extraction whereby powdered plant material is stirred with 70% methanol, filtered, and concentrated on a rotary evaporator. As this extraction is not selective, the crude aqueous extract contains contaminants co-extracted with fiavonol glycosides. In most cases, however, the impurities do not detract from the quality of the MALDI-MS results. 

When assessing the purity of dendrimers it should also be noted that NMR spectroscopic methods approach their limit of detection at contamination levels of ca. 5%. Additional chromatographic method such as gel permeation chromatography (GPC, SEC Section 7.1.2) or mass spectrometric methods (MALDI-MS, ESI-MS), as presented in Section 7.4, should also be employed in verification of structural perfection and purity of dendrimers. 

For successful analysis using MALDI, homogeneous sample preparation is critical. Many variables influence the preparation of a good MALDI sample, from the concentrations of the matrix and analyte, to the choice of matrix and the amount of internal energy the matrix imparts to the analyte during ionization, but may also include the hydrophobicity/hydrophilicity of the analyte, and the presence of contaminants such as detergents. 

The quality of the MALDI-TOF spectrum that will be obtained from the sample depends crucially on the sample/matrix preparation. The basic requirements are for a uniform microcrystalline layer of matrix/sample and the removal of salts and other contaminants. There are numerous published methods, but the one described here is robust and quite simple, requiring no pre-cleanup steps. For the best results, a high-quality matrix, as supplied by MALDI-MS manufac-... 

MALDI is relatively less sensitive to contamination by salts, buffers, detergents, and so on in comparison with other ionization techniques [41], The analyte must be incorporated into the matrix crystals. This process may generally serve to separate in solid phase the analyte from contaminants. However, high concentrations of buffers and other contaminants commonly found in analyte solutions can interfere with the desorption and ionization process of samples. Prior purification to remove the contaminants leads to improvements in the quality of mass spectra. For instance, the removal of alkali ions has proven to be very important for achieving high desorption efficiency and mass resolution. 

On-probe purification using derivatized MALDI probe surfaces has been described to simplify the sample preparation process. Various developments in this field have allowed the introduction of new techniques such as the surface-enhanced laser desorption ionization (SELDI) [42], The surface of the probe plays an active role in binding the analyte by hydrophobic or electrostatic interactions, while contaminants are rinsed away. In the same way, this technique uses targets with covalently coupled antibodies directed against a protein, allowing its purification from biological samples as urine or plasma. Subsequent addition of a droplet of matrix solution allows MALDI analysis. 

For both ESI and MALDI, the concentration of the sample and the complexity of the contaminants play important roles in both the sensitivity and the mass accuracy. Biological samples most often are diluted solutions of peptides or proteins containing a great number of contaminants. These two problems, dilution and contaminants, are not easy to handle, especially when the total amount of sample is low, such as picomoles. 

Generally, MALDI is more tolerant than ESI to many contaminants. This can in part be due in part to some separation occurring during the crystallization of the sample with the matrix [30], Whatever the ionization method, the quality of the mass spectrum is higher if the contamination is reduced. 

The widely used strategy for protein identification is depicted in Figure 8.15. This strategy thus is to cleave the protein either by trypsin, V8 protease, Lys-C endoprotease or by a reactant such as BrCN. The mixture then is analysed by mass spectrometry to obtain the molecular masses of the largest possible number of peptides. The two ionization methods, MALDI and ESI, are used. MALDI is best to use if one wants to avoid chromatographic separation, because it yields very simple spectra, has a better sensitivity and is not so sensitive to the presence of contaminants. However, ESI can be coupled directly with HPLC or capillary electrophoresis (CE) if a separation is wanted. Furthermore, ESI is often used with mass spectrometers that allow MS/MS data to be easily obtained. 

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