Omar Belgacem

Targeted profiling of atherogenic phospholipids in human plasma and lipoproteins of hyperlipidemic patients using MALDI-QIT-TOF-MS/MS

OBJECTIVES Phospholipids (PLs) are increasingly recognized as key molecules with potential diagnostic value in acute inflammation, CVD and atherosclerosis. We introduce a pioneer mass spectrometry (MS)-based approach aiming to investigate the relationship of specific plasma PL-subsets with atherogenic blood parameters in young patients with familial hyperlipidemia representing high-CVD-risk groups. METHODS Plasma of carefully phenotyped FH and FCH patients as well as normolipidemic subjects (age 13 ± 5 years, n = 20) was used. Clinical parameters were assessed using standard laboratory techniques and lipids were subjected to a direct targeted monitoring using LC-ESI-SRM- and MALDI-QIT-TOF-MS/MS, respectively. Statistical analysis was performed to evaluate correlations between PL data and the clinical parameters. RESULTS Most characteristically significant differences of SM/PC and PC/LPC ratios and positive correlations between SM vs. LDL-C (r = 0.946; p = 0.004) and LPC vs. VLDL-C (r = 0.669; p = 0.218) were observed in FH in contrast to the other study groups. OxPC levels were found in the range of ∼2-20 μmol/L with predominance of short-chain aldehydic species (e.g. SOVPC). A positive correlation of OxPCs with IMT (r = 0.952; p = 0.052) and HDL-C (r = 0.893; p = 0.016) but negative correlation with OxLDL (r = -0.910; p = 0.096) was observed. CONCLUSIONS Our study was a first attempt to use a MALDI-QIT-TOF-MS/MS based clinical lipidomics approach to investigate atherogenic dyslipidemia in young patients with familial hyperlipidemia. This technique represents a promising platform for clinical screening of lipid biomarkers in the future.

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 linear TOF mass spectrometry of PEGylated (glyco)proteins.

PEGylation of proteins is a fast growing field in biotechnology and pharmaceutical sciences owing to its ability to prolong the serum half-life time of recombinant proteins. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) has been shown to be a powerful tool in the analysis of several PEGylated small proteins. Here we present data obtained with a standard secondary electron multiplier (SEM) and a high mass (HM) detector combined with a MALDI linear TOF MS system for the detection of PEGylated (glyco)proteins in the range of 60-600 kDa. Examples of MALDI TOF MS of small (interferon alpha2a), middle (human serum albumin (HSA)) and high molecular mass proteins (coagulation factor VIII and von Willebrand factor (vWF), both heavily glycosylated proteins) are presented. The particular challenge for the analysis was the heterogeneity of the (glyco)proteins in the high molecular weight range in combination with additional PEGylation, which even introduced more heterogeneity and was more challenging for interpretation. Nevertheless, the performance of MALDI linear TOF MS with both detector systems in terms molecular weight and heterogeneity determination depending on the m/z range was superior to the other methods. Although the SEM was able to obtain information about protein PEGylation in the mass range up to 100 kDa (e.g. PEGylated HSA), the HM system was crucial for detection of HM ions (e.g. PEGylated recombinant vWF), which was impossible with the standard SEM.

Glycation pattern of peptides condensed with maltose, lactose and glucose determined by ultraviolet matrix‐assisted laser desorption/ionization tandem mass spectrometry

Protein glycation is the non-enzymatic condensation of sugars with proteins. Although commonly occurring in both the therapeutic and food/beverage industries, protein glycation has not been the focus of many proteomic investigations. This study aims to establish a reliable mass spectrometric method for screening large tandem mass spectrometric (MSMS) datasets for protein glycation with glucose, lactose and maltose. Control experiments using a standard peptide containing a single glycation site led to the discovery of characteristic neutral loss fragmentation patterns in MSMS analysis for glucose, lactose and maltose condensed with peptides. Valid in both tandem time-of-flight (TOFTOF) and quadrupole ion trap time-of-flight matrix-assisted laser desorption/ionization (QIT TOF MALDI) mass spectrometers, these neutral loss signatures were then applied to elucidation of modified peptides from a complex human serum albumin (HSA) digest glycated with each of the proposed sugars. Screening of these large datasets was made possible by specifically designed software solutions that enable the input of detailed user-defined post-translational modifications that are not included in the universally available databases such as Unimod.

In-source decay and pseudo tandem mass spectrometry fragmentation processes of entire high mass proteins on a hybrid vacuum matrix-assisted laser desorption ionization-quadrupole ion-trap time-of-flight mass spectrometer.

In-source decay (ISD), although a process known for decades in mass spectrometry, has a renewed interest due to increased theoretical knowledge in fragmentation processes of large biomolecules coupled with technological improvements. We report here an original method consisting of isolating matrix-assisted laser desorption ionization (MALDI)-generated in-source fragments of large proteins and subsequently performing selective fragmentation experiments (up to four cycles) using a hybrid MALDI quadrupole ion-trap time-of-flight mass spectrometer (MALDI-QIT-TOF). This technology takes advantage of keeping high resolution on the selection of precursors and detection of fragments. It allows exhaustive N- and C-terminal sequencing of proteins. In this work, human serum albumin (HSA), β-casein, and recombinant Tau proteins were submitted to in source decay in the MALDI source. The fragments were stored in the ion-trap and submitted to sequential collision-induced dissociation (CID). Finally, ISD and pseudo MS(n) were performed on oxidized Tau protein and acetylated bovine serum albumin to identify amino acid modifications. This work highlights the potential of the MALDI-QIT-TOF instrument for pseudo MS(n) strategies and top down proteomics.

Influence of HSA and IgG on LDL oxidation studied by size-exclusion chromatography and phospholipid profiling using MALDI tandem-mass spectrometry.

In the present study a direct detection approach combining size-exclusion chromatography (SEC) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem-mass spectrometry (MALDI-QIT-TOF-MS/MS) was applied to investigate the influence of HSA and IgG on LDL oxidation in vitro. SEC analysis showed an increase of protein aggregation during LDL-oxidation that could be essentially suppressed in the presence of HSA. In parallel, lipid peroxidation measured by TBARS assay over 24h was inhibited by 95-100% in the presence of HSA but only 0-34% by IgG, respectively. MALDI phospholipid profiles showed considerable decrease of signals from PCs containing sn-2 PUFAs (18:2 or 20:4) accompanied by increase of sn-2 LPCs indicating for specific breakdown of PUFA-containing PLs during LDL-oxidation. These effects were nearly 100% inhibited in the presence of HSA but not by IgG, respectively. Among known pro-atherogenic PL species present in human plasma sphingomyelin (SM16:0) was bound in significant amounts to HSA but not IgG after incubation with oxLDL. Moreover, our investigation showed that LPCs containing SAFAs (16:0 or 18:0) were specifically bound to HSA, while those containing PUFAs (18:2 and 18:3) were preferentially associated with IgG. In summary, the presented methodology provides a promising platform for studying lipid-protein interactions in vivo.

Axial spatial distribution focusing: improving MALDI-TOF/RTOF mass spectrometric performance for high-energy collision-induced dissociation of biomolecules.

Rationale For the last two decades, curved field reflectron technology has been used in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometers, assisting in the generation of post-source-decay (PSD) or collision-induced dissociation (CID) without decelerating precursor ions, producing true high-energy CID spectra. The result was the generation of product ion mass spectra with product ions typical of high-energy (10 keV and beyond) collision processes. The disadvantage of this approach was the lack of resolution in CID spectra resulting from the excess laser energy deposition used to generate those MS/MS spectra. The work presented in this study overcomes this limitation and includes comprehensive examples of high-energy and high-resolution CID MALDI-MS/MS spectra of biomolecules. Methods The devices used in this study are TOF/RTOF instruments equipped with a high-vacuum MALDI ion source. High-resolution and high-energy CID spectra result from the use of axial spatial distribution focusing (ASDF) in combination with curved field reflectron technology. Results A CID spectrum of the P14R1 peptide exhibits product ion resolution in excess of 10,000 (FWHM) but at the same time yields typical high-energy product ions such as w- and [y–2]-type ion series. High-energy CID spectra of lipids, exemplified by a glycerophospholipid and triglyceride, demonstrate C–C backbone fragmentation elucidating the presence of a hydroxyl group in addition to double-bond positioning. A complex high mannose carbohydrate (Man)8(GlcNAc)2 was also studied at 20 keV collision energy and revealed further high-energy product ions with very high resolution, allowing unambiguous detection and characterization of cross-ring cleavage-related ions. Conclusions This is the first comprehensive study using a MALDI-TOF/RTOF instrument equipped with a curved field reflectron and an ASDF device prior to the reflectron.