Michaela Scigelova

Coupling liquid chromatography to Orbitrap mass spectrometry.

The Orbitrap mass analyzer has become a mainstream mass spectrometry technique. In addition to providing a brief introduction to the Orbitrap technology and its continuing development, this article reviews the most recent publications quoting the use of the Orbitrap detection for a variety of chromatographic separation techniques. Its coupling to reversed-phase liquid chromatography (LC) represents undoubtedly the most ubiquitous approach to both small molecule and proteomic analyses. Multi-dimensional LC separations have an important role to play in the proteomics applications while an ultra-high-pressure LC is more frequently encountered in the area of metabolomics and metabolite analysis. Recently, special chromatographic techniques such as hydrophilic interaction chromatography and its variations have also been also cited with the Orbitrap detection.

Fourier transform mass spectrometry

This article provides an introduction to Fourier transform-based mass spectrometry. The key performance characteristics of Fourier transform-based mass spectrometry, mass accuracy and resolution, are presented in the view of how they impact the interpretation of measurements in proteomic applications. The theory and principles of operation of two types of mass analyzer, Fourier transform ion cyclotron resonance and Orbitrap, are described. Major benefits as well as limitations of Fourier transform-based mass spectrometry technology are discussed in the context of practical sample analysis, and illustrated with examples included as figures in this text and in the accompanying slide set. Comparisons highlighting the performance differences between the two mass analyzers are made where deemed useful in assisting the user with choosing the most appropriate technology for an application. Recent developments of these high-performing mass spectrometers are mentioned to provide a future outlook.

Electrospray ionization mass spectrometry identifies substrates and products of lipoprotein-associated phospholipase A2 in oxidized human low density lipoprotein

There is increasing evidence that modified phospholipid products of low density lipoprotein (LDL) oxidation mediate inflammatory processes within vulnerable atherosclerotic lesions. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is present in vulnerable plaque regions where it acts on phospholipid oxidation products to generate the pro-inflammatory lysophsopholipids and oxidized non-esterified fatty acids. This association together with identification of circulating Lp-PLA2 levels as an independent predictor of cardiovascular disease provides a rationale for development of Lp-PLA2 inhibitors as therapy for atherosclerosis. Here we report a systematic analysis of the effects of in vitro oxidation in the absence and presence of an Lp-PLA2 inhibitor on the phosphatidylcholine (PC) composition of human LDL. Mass spectrometry identifies three classes of PC whose concentration is significantly enhanced during LDL oxidation. Of these, a series of molecules, represented by peaks in the m/z range 594-666 and identified as truncated PC oxidation products by accurate mass measurements using an LTQ Orbitrap mass spectrometer, are the predominant substrates for Lp-PLA2. A second series of oxidation products, represented by peaks in the m/z range 746-830 and identified by LTQ Orbitrap analysis as non-truncated oxidized PCs, are quantitatively more abundant but are less efficient Lp-PLA2 substrates. The major PC products of Lp-PLA2, saturated and mono-unsaturated lyso-PC, constitute the third class. Mass spectrometric analysis confirms the presence of many of these PCs within human atherosclerotic lesions, suggesting that they could potentially be used as in vivo markers of atherosclerotic disease progression and response to Lp-PLA2 inhibitor therapy.

Advances in bioanalytical LC–MS using the Orbitrap™ mass analyzer

The continuing desire to analyze complex biological samples with the minimum number of steps places high demands on increasing speed, dynamic signal range, quantitative capability and the facility with which the mass spectrometers can interface with chromatographic separation methods. Reliable identification of metabolites in complex mixtures requires robust mass spectrometers with high resolving power, mass accuracy, sensitivity and dynamic range, while tandem MS is an invaluable tool for further structural characterization. This review begins with a discussion of the key properties of the Orbitrap™ mass analyzer: mass accuracy, resolution, fidelity of isotope pattern abundancies and dynamic range. The main objective is to provide an overview of Orbitrap applications in the field of bioanalysis. Specific areas of drug metabolism, doping control and food contaminants are discussed in detail illustrating the performance and versatility of the Orbitrap mass analyzer.

Identification of synaptic plasma membrane proteins co‐precipitated with fibrillar β‐amyloid peptide

The β‐amyloid peptide that is overproduced in Alzheimers disease rapidly forms fibrils, which are able to interact with various molecular partners. This study aimed to identify abundant synaptosomal proteins binding to the fibrillar β‐amyloid (fAβ) 1–42. Triton X‐100‐soluble proteins were extracted from the rat synaptic plasma membrane fraction. Interacting proteins were isolated by co‐precipitation with fAβ, or with fibrillar crystallin as a negative control. Protein identification was accomplished (1) by separating the tryptically digested peptides of the protein pellet by one‐dimensional reversed‐phase HPLC and analysing them using an ion‐trap mass spectrometer with electrospray ionization; and (2) by subjecting the precipitated proteins to gel electrophoretic fractionation, in‐gel tryptic digestion and to matrix‐assisted laser desorption/ionization time‐of‐flight mass measurements and post‐source decay analysis. Six different synaptosomal proteins co‐precipitated with fAβ were identified by both methods: vacuolar proton‐pump ATP synthase, glyceraldehyde‐3‐phosphate dehydrogenase, synapsins I and II, β‐tubulin and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase. Most of these proteins have already been associated with Alzheimers disease, and the biological and pathophysiological significance of their interaction with fAβ is discussed.

Quantitative assessment of the contribution of high resolution mass spectrometric analysis to the reliability of compound confirmation

Applications of high resolution mass spectrometry (HRMS) in food safety and residue analysis have increased remarkably over the last few years. The high resolution detection of ions reportedly enhances the assay selectivity but quantitative assessment of HRMS contribution to the assay selectivity has not yet been undertaken. We devised a method to assess the impact of instrument resolution on the probability that a spectral assignment to a given compound was made in error. The method allows for evaluating the quality of a spectral assignment based on resolution and the number of fragmentation stages. It thus provides a firm basis for comparing analytical methods performed on very different mass spectrometric instrumental platforms as well as in the context of the current regulatory framework.

Quantitative analysis of caveolin-rich lipid raft proteins from primary and metastatic colorectal cancer clones.

Caveolin-rich lipid rafts (CLRs) are thickened sections of the cell membrane that are composed of the integral membrane proteins caveolins together with saturated long chain fatty acids, cholesterol and lipids. Membrane proteins - lipid raft proteins in particular - may play important roles in cell signaling and cell-cell interaction. Due to their unique structure, CLRs seem to be the preferred docking site for specific proteins involved in focal adhesion and cancer metastasis. Our objective was thus to identify and quantify CLR proteins from primary and metastatic colorectal cancer (CRC) clones. We found differential expression of nine CLR proteins from primary and metastatic CRC clones. Among the identified proteins, an immune system inhibiting protein was significantly overexpressed in the metastatic clone, while cell adhesion and transport molecules were among the overexpressed proteins in the primary clone. All the identified CRL proteins are involved in tumorigenesis, specifically metastasis, and may thus serve as therapeutic targets. A novel concept for identification and quantification of CLR proteins with label-free mass spectrometry method was specifically examined in this study. Validation of the method against immunoblotting and FACS analysis indicates that it can be applied for the identification of novel biomarkers for cancer and metastasis.

Detailed Functional and Proteomic Characterization of Fludarabine Resistance in Mantle Cell Lymphoma Cells

Mantle cell lymphoma (MCL) is a chronically relapsing aggressive type of B-cell non-Hodgkin lymphoma considered incurable by currently used treatment approaches. Fludarabine is a purine analog clinically still widely used in the therapy of relapsed MCL. Molecular mechanisms of fludarabine resistance have not, however, been studied in the setting of MCL so far. We therefore derived fludarabine-resistant MCL cells (Mino/FR) and performed their detailed functional and proteomic characterization compared to the original fludarabine sensitive cells (Mino). We demonstrated that Mino/FR were highly cross-resistant to other antinucleosides (cytarabine, cladribine, gemcitabine) and to an inhibitor of Bruton tyrosine kinase (BTK) ibrutinib. Sensitivity to other types of anti-lymphoma agents was altered only mildly (methotrexate, doxorubicin, bortezomib) or remained unaffacted (cisplatin, bendamustine). The detailed proteomic analysis of Mino/FR compared to Mino cells unveiled over 300 differentially expressed proteins. Mino/FR were characterized by the marked downregulation of deoxycytidine kinase (dCK) and BTK (thus explaining the observed crossresistance to antinucleosides and ibrutinib), but also by the upregulation of several enzymes of de novo nucleotide synthesis, as well as the up-regulation of the numerous proteins of DNA repair and replication. The significant upregulation of the key antiapoptotic protein Bcl-2 in Mino/FR cells was associated with the markedly increased sensitivity of the fludarabine-resistant MCL cells to Bcl-2-specific inhibitor ABT199 compared to fludarabine-sensitive cells. Our data thus demonstrate that a detailed molecular analysis of drug-resistant tumor cells can indeed open a way to personalized therapy of resistant malignancies.