Gerald Stübiger

Analysis of Oxidized Phospholipids by MALDI Mass Spectrometry Using 6-Aza-2-thiothymine Together with Matrix Additives and Disposable Target Surfaces

6-Aza-2-thiothymine (ATT) is introduced as novel matrix system for the analysis of oxidized phospholipids (OxPLs) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). A systematic evaluation comparing different established and novel matrix substances, especially 2,4,6-THAP matrix (Stubiger, G.; Belgacem O. Anal. Chem. 2007, 79, 3206-3213) as reference compound for phospholipid analysis, and specific matrix additives was performed. Thereby, ATT turned out to be the reagent of choice for MALDI analysis of major biologically relevant OxPL classes (e.g., OxPC, OxPE, and OxPS) in positive and negative ionization mode. ATT used together with specific chaotropic reagents at low concentration (0.5-2 mM) acting as OxPL ionization enhancers revealed an excellent comatrix system for application with MALDI instrument types employing UV- and Nd:YAG laser systems (337 and 355 nm). Moreover, disposable MALDI targets surfaces with specific physicochemical properties (e.g., metallized glass or polymeric substrates) were revealed as superior over stainless steel in terms of reduced chemical background noise ( approximately 10-fold better S/N ratios), increased mass spectral reproducibility, and enhanced sensitivity (LOD approximately 250-500 fg on target). The combination of these parameters offers a significant advantage for highly sensitive OxPL profiling by MALDI-MS of biological samples (e.g., human plasma) at trace levels.

Analysis of human plasma lipids and soybean lecithin by means of high‐performance thin‐layer chromatography and matrix‐assisted laser desorption/ionization mass spectrometry

An improved analytical strategy for the analysis of complex lipid mixtures using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in combination with high-performance thin-layer chromatography (HPTLC) is reported. Positive ion MALDI RTOF MS was applied as a rapid screening tool for the various neutral (e.g. triacylglycerols) and polar (e.g. glycerophospholipids and -sphingolipids) lipid classes derived from crude lipid extracts of e.g. human plasma as well as soybean lecithin. Finally, MALDI seamless post-source decay (PSD) product ion analysis was performed in order to obtain further structural information (head- and acyl-group identification) of selected lipid species and structure verification. A Coomassie Brilliant Blue R-250 staining protocol for lipids on HPTLC plates was evaluated and was found to be fully compatible with subsequent MALDI-MS. Lipids were analyzed after elution from the HPTLC phase material of the selected band (corresponding to certain lipid classes) by using the proper organic solvent mixture or in few cases directly from the HPTLC plates (a type of on-line HPTLC/MALDI-MS coupling). More than 70 distinct lipid species from seven different lipid classes in the range between m/z 500 and 1500 could be identified from the lipid extracts of human plasma and soybean lecithin, respectively. The general high sensitivity of MALDI-MS detection allowed the analysis of even minor lipid classes from only very small volumes of human plasma (50 microL). The combination of HPTLC, Coomassie staining and positive ion MALDI curved field RTOF-MS represents a straightforward strategy during lipidomics studies of food and clinically relevant human lipid samples.

MALDI Seamless Postsource Decay Fragment Ion Analysis of Sodiated and Lithiated Phospholipids

In this paper, we present the results of a detailed study using MALDI seamless postsource decay (sPSD) fragment ion analysis of all major glycerophospholipid (GPL) classes, cardiolipin (bisphosphatidylglycerol), and sphingomyelin, respectively. The matrix compound 2,4,6-trihydroxyacetophenon recently introduced for lipid analysis (Stübiger, G.; Belgacem O. Anal. Chem. 2007, 79, 3206-3213) was applied in conjunction with a high-resolution curved field reflectron analyzer allowing detection of the fragment ions without stepping the reflectron voltages (seamless PSD). This instrumental feature helps to define in a fast way the polar headgroups of the different GPL classes and gives information about the constituent fatty acid residues dependent on the type of adduct ion used. Our experiments demonstrate that fragment ions specifying the fatty acid composition of the lipid molecules could only be generated from cationized molecular ions (sodiated or lithiated). Additionally, information about the stereospecificity of the fatty acid residues on the glycerol backbone (sn-1, and -2 position) of particular GPLs could be obtained during sPSD analysis. In the case of phosphatidylcholine, significant fragmentation related to the loss of the acyl groups could only be observed from [M + Li](+) ions. Generally, alkali adduction (sodium and lithium) enhanced fragmentation of most lipid classes, particularly favoring fragment ions associated with the polar headgroups.