Matrix-assisted laser desorption/ionisation technique

Jackson AT, Bunn A, Chisholm MS. Utihsing matrix-assisted laser desorption/ionisation techniques for the generation of stractural information from different end-group function-ahsed poly(methyl methacrylate)s. Polymer 2008 49 5254-5261. 

As evident from Scheme 7.13, most modern ionisation techniques have been used for TLC-MS, and no single ionisation method is used exclusively with TLC-MS. Various ionisation methods may be applied that avoid the need to evaporate the sample into an El or Cl source these are based in particular on sputtering (FAB, SIMS) or laser desorption. Several sputtering methods of ionisation do not require the use of a liquid matrix, e.g. TLC-SIMS [797], Recent developments include the use of matrix-assisted laser desorption ionisation (MALDI) and surface-assisted laser desorption ionisation (SALDI). It is obvious that TLC-MS is complemented with TLC-MS11 [800] and TLC-HRMS techniques. Table 7.82 lists the general characteristics of TLC-MS. 

Experimental considerations Sample preparation and data evaluation are similar to membrane osmometry. Since there is no lower cut-off as in membrane osmometry, the method is very sensitive to low molar mass impurities like residual solvent and monomers. As a consequence, the method is more suitable for oligomers and short polymers with molar masses up to (M)n 50kg/mol. Today, vapour pressure osmometry faces strong competition from mass spectrometry techniques such as matrix-assisted laser desorption ionisation mass spectrometry (MALDI-MS) [20,21]. Nevertheless, vapour pressure osmometry still has advantages in cases where fragmentation issues or molar mass-dependent desorption and ionization probabilities come into play. 

Mass spectrometry is used to identify unknown compounds by means of their fragmentation pattern after electron impact. This pattern provides structural information. Mixtures of compounds must be separated by chromatography beforehand, e.g. gas chromatography/mass spectrometry (GC-MS) because fragments of different compounds may be superposed, thus making spectral interpretation complicated or impossible. To obtain complementary information about complex mixtures as a whole, it may be advantageous to have only one peak for each compound that corresponds to its molecular mass ([M]+). Even for thermally labile, nonvolatile compounds, this can be achieved by so-called soft desorption/ionisation techniques that evaporate and ionise the analytes without fragmentation, e.g. matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). 

While fast atom bombardment (FAB) [66] and TSI [25] built up the basis for a substance-specific analysis of the low-volatile surfactants within the late 1980s and early 1990s, these techniques nowadays have been replaced successfully by the API methods [22], ESI and APCI, and matrix assisted laser desorption ionisation (MALDI). In the analyses of anionic surfactants, the negative ionisation mode can be applied in FIA-MS and LC-MS providing a more selective determination for these types of compounds than other analytical approaches. Application of positive ionisation to anionics of ethoxylate type compounds led to the abstraction of the anionic moiety in the molecule while the alkyl or alkylaryl ethoxylate moiety is ionised in the form of AE or APEO ions. Identification of most anionic surfactants by MS-MS was observed to be more complicated than the identification of non-ionic surfactants. Product ion spectra often suffer from a reduced number of negative product ions and, in addition, product ions that are observed are less characteristic than positively generated product ions of non-ionics. The most important obstacle in the identification and quantification of surfactants and their metabolites, however, is the lack of commercially available standards. The problems with identification will be aggravated by an absence of universally applicable product ion libraries. 

Below we report methodological studies based upon HPLC, GC/FID, GC-MS, LC-MS, matrix-assisted laser desorption ionisation coupled with time-of-flight mass spectrometry (MALDI-ToF/MS), CE, proton nuclear magnetic resonance ( I INMR), RIA and enzymatic colorimetric techniques. 

The mass spectrometry analysis was performed by the matrix assisted laser desorption/ionisation time-of-flight (S8-MALDI) technique using a Voyager-DE PRO Biospectrometry Workstation (Applied Biosystems, USA). Radiation pulses of 0.5 ns and 3 Hz frequency from N2 laser operating at 337 nm were used to desorb the species and negative/positive ions formed were detected in reflectron mode. Sulfur used as a matrix material was also dissolved in toluene and mixed with the samples solution prior to deposition onto a target. 

Two techniques that have become preffered for ionisation of proteins/peptides is electrospray ionisation (ESI) and matrix-assisted laser desorption/ionisation (MALDI). Although different combinations of ionisation techniques and mass analyser exist, MALDI usually is coupled with a time-of-flight (TOF) (Figure 7) tube as a mass analyser while ESI is tradionally combined with quadrupole mass analysers. Instruments capable of MS/MS have the ability to select ions of particular m/z ratio from a mixture, to fragment selected ions and to record the precise masses of the resulting fragment ions. If this process is applied to the analysis of peptide ions, in principle the amino acid sequence of the peptide can be deduced. 

Mass spectrometry has assumed great importance in determinations of the molar masses of biological macromolecules, even quite large ones. This is due to developments such as electrospray ionisation (ESI) and matrix assisted laser desorption/ ionisation (MALDI), which have made it possible to determine the molar masses of biopolymers up to several 100 kDa (Pitt 1996 Kellner et al. 1999 Snyder 2000). The combination of MALDI techniques with time-of-flight mass spectrometers (MALDI-TOF) is of particular significance for determination of the molar masses of proteins with high sensitivity (typically pmol quantities, although exceptionally fmol) and precision (proteins up to 100 kDa with precision of about 0.01 %). Mass spectrometry can provide very accurate measurements of protein molar mass that can yield information about even minor structural modifications not readily accessible by other means. 

The classical area of application of mass spectrometry has been with small volatile compounds, although non-volatile samples could be analysed if they were suitably derivatised. The application of mass spectrometry to large complex molecules like proteins has been made possible by the development of novel ionisation techniques which enable large molecules (> 200 kDa) to be introduced into the mass spectrometer in an intact form suitable for analysis (Siuzdak 1996 Dass 2000). Of the various techniques that have been developed, electrospray ionisation (ESI) and matrix-assisted laser desorption ionisation (MALDI) are the ones best suited for use with macromolecules and macromolecular complexes. 

Larhrib, H. Wells, M.H. Rubinstein, M.H., et al. Characterization of PEGs using matrix-assisted laser desorption/ ionisation mass spectrometry and other related techniques. Int. J. Pharm. 1997, 14, 187-198. 

Chemical ionisation mass spectra (CIMS NHJ as ionisation reactant) can yield satisfactory spectra from underivatised peptides and field-desorption ionisation has been shown to give intense [M+ 1]+ ions from otherwise involatile peptide derivatives (for example, CH3—CO—Gly—Arg—Arg—Gly—OCH3 Buehler el al., 1974), but less sequence information is gained in these mild ionisation procedures because less fragmentation occurs and there are relatively few peaks in the mass spectrum. MALDF - matrix-assisted laser desorption ionisation MS - is an acronym that is encountered in recent literature for this ionisation technique. 

Matrix assisted laser desorption ionisation (MALDI) is an important technique for characterisation and fingerprinting of large molecules. However, solvents used at sample preparation have a negative effect on the quahty of the fingerprint. Work is therefore performed on development of solvent free sample preparation methods for characterisation of synthetic polymers [61]. 

Matrix-assisted laser desorption ionisation (MALDI) can analyse molecules with large mass >10 000 Da. The technique is similar to FAB and is also considered a soft ionisation process. The analyte is mixed with a matrix such as sinapinic acid or 2,5-dihydroxybenzoic acid that strongly absorbs UV radiation. A few microlitres of this analyte/matrix mixture is deposited on the sample plate and dried. The plate is inserted into the source region... 

The mass spectrometry of oligonucleotides is a rapidly expanding research area dominated by the techniques of electrospray ionisation and matrix assisted laser desorption-ionisation, time of flight (MALDI-TOF) mass spectrometry. A number of reviews of this area have been published this year. Several... 

A reliable analysis of multicomponent mixtures is particularly difficult and the situation becomes more complicated and less meaningful with increasing mixture size. Nevertheless, the analysis of peptide libraries is possible by mass spectroscopic means.366-572 mixture size increases, the main aim of any analysis is to ensure that the vast majority of expected library members are represented in the mixture. The use of MS in conjunction with other analytical techniques (HPLC-MS, GC-MS, MS-MS) have been used successfully in library characterisation. 3.425.573 Electronspray-MS is a particularly mild method for ionisation. s Matrix-assisted Laser Desorption Ionisation - time-of-flight (MALDI-TOF) mass spectrometry is a tool of increasing importance in multicomponent analysis. Its reduced tendency to preferential ionisation results in an increased likelihood of observing ions from all components.5 575... 

The sample molecules can be ionised by one of several techniques. In electron impact ionisation, (El), electrons are fired at the sample molecules, whereas in chemical ionisation, (Cl), the sample molecules are collided with a reactive gas. The sample can also be bombarded with argon atoms (fast atom bombardment, FAB) or the dissolved sample can be sprayed into an electric field electrospray ionisation, ESI). Furthermore, the sample can be co-crystallised with a matrix and then ions can be generated by exposure to photons (matrix assisted laser desorption ionisation, MALDI). 

Samperi and co-workers [60] examined the thermal degradation behaviour of PET under nitrogen within the temperature range used in commercial processing of this polymer (270-370 °C) using a combination of matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry and nuclear magnetic resonance techniques. 

The sensitive requirements for exposure assessment have been met by the recent development of mass spectrometry (MS) techniques for the characterisation of proteins expressed by a genome, tissue or cell. Of particular interest is the matrix-assisted laser-desorption/ionisation (MALDI), which makes it possible to obtain protein mass fingerprinting for a wide range of proteins by MS. This method involves selectively cutting proteins by enzymatic actions, and comparing the fragment masses with theoretical peptides available in bio-informatic databases. 

In the early nineties nuclear magnetic resonance (NMR) spectroscopy was the preferred technique in order to fully characterise structures of oligosaccharides. Nowadays, both matrix assisted laser desorption/ ionisation time-of-flight (MALDI-TOF) and electrospray MS have become routine and powerful techniques in the identification of structural features of oligosaccharides (26-28), even to determine the positions of residues within oligosaccharides (29,30). 

The different stages of the preparation of peptide surfaces can be confirmed with surface-sensitive physical and chemical analysis techniqnes. For gold-based SAMs, Mrksich and co-workers have introduced a matrix-assisted laser desorption ionisation time-of-ftight (MALDI-TOF) mass spectrometry-based analysis procedure with which they are able to identify the presence of various surface functional groups via their mass (Yeo Mrksich, 2006 Yeo et al., 2003). Although this method is applicable to SAMs, it is not strictly a surface sensitive technique, as the desorption process in MALDI is not confined to the uppermost layer of a material. 

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