| Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is a powerful analytical tool available for studying different biomolecules and their complex mixtures. Special consideration for sample handling, preparation techniques and subsequent analysis by MALDI MS must be taken in order to guarantee the best analysis possible in terms of accuracy and reproducibility of analyte identification, relative and absolute quantification, and the number of mixture constituents detected and characterized. The use of various compounds as matrix additives was found to enhance ion signal intensities of the analyte biomolecules, in particular carbohydrates, over the conventionally used matrix materials. Aniline and N,N-dimethylaniline were identified as effective additives to 2,5-dihydroxybenzoic acid, a widely used MALDI matrix, for superior analysis of carbohydrates. Perhaps the most important advantages of using these materials is the fact that sample oligosaccharides need not be derivatized, thus making highly sensitive analysis of native glycans attainable by MALDI MS. Use of these materials also offers some unique advantages, such as a more reliable and reproducible quantitative analysis of sugars and a possibility for designing efficient workflows for automated detection of oligosaccharides in complex biological samples. Multidimensional separation procedures, involving various types of liquid chromatography and capillary electrophoresis, were employed for targeted sample fractionation according to modifications of interest, as well as to reduce the signal suppression of the less abundant analytes and to improve the resolution of the sample mixture components. Capillary electrophoresis was demonstrated to be an effective technique for isolating glycopeptides containing sialic acids. Cellulose-based solid phase extraction procedures were found to be extremely useful and efficient for isolation of glycopeptides, and were shown to offer real practical benefits for routine glycoproteomic and glycomic applications. Reversed-phase chromatography was proven to be essential in oligosaccharide purification and in site-specific analysis of protein glycosylation. Examples of the aforementioned techniques and methods with respect to their application to various controlled standard systems, as well as real-life precious biological samples are presented in this work. |
