Stephen Macha

Influence of ionization energy on charge-transfer ionization in matrix-assisted laser desorption/ionization mass spectrometry

Abstract In this study, non-polar matrices are used in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to analyze selected non-polar analytes. Our hypothesis is that gas-phase charge-transfer reactions between matrix and analyte are responsible for the generation of analyte radical molecular ions. Following this hypothesis, the ionization energies of the matrices and analytes should have a direct influence on the production of radical molecular cations of the analytes. Several non-polar analytes, including ferrocene and ferrocene derivatives, trans-stilbene, triphenylphosphine, 2,2′-methylenebis(6-tert-butyl-4-methylphenol), biphenyl and 1,4-bis(methylthio)benzene were studied using positive-ion mode MALDI-TOFMS. The results of these studies demonstrate that formation of the radical molecular cation depends on the difference in ionization energies between the matrix and the analyte. The propensity for charge-transfer ionization, as opposed to proton-transfer ionization, for these analytes, was confirmed using atmospheric pressure chemical ionization mass spectrometry. Charge-transfer ionization using non-polar matrices in MALDI-MS is a suitable method for the characterization of a number of non-polar, thermally labile analytes.

Silver cluster interferences in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of nonpolar polymers

Potential difficulties associated with background silver salt clusters during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of nonpolar polymers are reported. Silver salt cluster ions were observed from m/z 1500 to 7000 when acidic, polar matrices, such as 2,5-dihydroxybenzoic acid (DHB), all-trans-retinoic acid (RTA) or 2-(4-hydroxyphenylazo)benzoic acid (HABA), were used for the analysis of nonpolar polymers. These background signals could be greatly reduced or eliminated by the use of nonpolar matrices such as anthracene or pyrene. Representative examples of these background interferences are demonstrated during the analysis of low molecular weight nonpolar polymers including polybutadiene and polystyrene. Nonpolar polymers analyzed with acidic, polar matrices (e.g., RTA) and silver cationization reagents can yield lower quality mass spectral results when interferences due to silver clusters are present. Replacing the polar matrices with nonpolar matrices or the silver salts with copper salts substantially improved the quality of the analytical results. In addition, it was found that silver contamination cannot be completely removed from standard stainless steel sample plates, although the presence of silver contamination was greatly reduced after thorough cleaning of the sample plate with aluminum oxide grit. Carry-over silver may cationize polymer samples and complicate the interpretation of data obtained using nonpolar matrices in the absence of added cationization reagents.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of polymers

Matrix-assisted laser desorption/ionization mass spectrometry has been demonstrated to be a powerful analytical technique for the analysis of polymeric materials. The advantages of this technique for such analyses include low sample consumption, ease of sample preparation, short analysis times, and soft ionization which leads to negligible or no fragmentation of analytes. It provides absolute, fast and accurate molecular masses for polymers with narrow polydispersity as opposed to relative masses provided by other techniques. It provides masses for the entire polymer distribution instead of the average value, hence providing molecular mass information which can be used to obtain the mass of the end-groups, mass of the repeat unit (monomer), and chemical modifications on the polymer if oligomer resolution is attained. This review concentrates on the developments in methodology that have allowed for the increased use of this technique for polymer analysis.

Application of Nonpolar Matrices for the Analysis of Low Molecular Weight Nonpolar Synthetic Polymers by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

The application of nonpolar matrices for the analysis of low molecular weight nonpolar synthetic polymers using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is demonstrated. Anthracene, pyrene, and acenaphthene were utilized as nonpolar matrices for the analysis of polybutadiene, polyisoprene, and polystyrene samples of various average molecular weights ranging from about 700 to 5000. The standard MALDI-MS approach for the analysis of these types of polymers involves the use of conventional acidic matrices, such asall-trans-retinoic acid, with an additional cationization reagent. The nonpolar matrices used in this study are shown to be as equally effective as the conventional matrices. The uniform mixing of the nonpolar matrices and the nonpolar analytes enhances the MALDI-MS spectral reproducibility. Silver salts were found to be the best cationization reagents for all of the cases studied. Copper salts worked well for polystyrene, poorly for polyisoprene, and not at all for polybutadiene samples. These matrices should be useful for the characterization of hydrocarbon polymers and other analytes, such as modified polymers, which may potentially be sensitive to acidic matrices.

Speciated human high-density lipoprotein protein proximity profiles

It is expected that the attendant structural heterogeneity of human high-density lipoprotein (HDL) complexes is a determinant of its varied metabolic functions. To determine the structural heterogeneity of HDL, we determined major apolipoprotein stoichiometry profiles in human HDL. First, HDL was separated into two main populations, with and without apolipoprotein (apo) A-II, LpA-I and LpA-I/A-II, respectively. Each main population was further separated into six individual subfractions using size exclusion chromatography (SEC). Protein proximity profiles (PPPs) of major apolipoproteins in each individual subfraction was determined by optimally cross-linking apolipoproteins within individual particles with bis(sulfosuccinimidyl) suberate (BS(3)), a bifunctional cross-linker, followed by molecular mass determination by MALDI-MS. The PPPs of LpA-I subfractions indicated that the number of apoA-I molecules increased from two to three to four with an increase in the LpA-I particle size. On the other hand, the entire population of LpA-I/A-II demonstrated the presence of only two proximal apoA-I molecules per particle, while the number of apoA-II molecules varied from one dimeric apoA-II to two and then to three. For most of the PPPs described above, an additional population that contained a single molecule of apoC-III in addition to apoA-I and/or apoA-II was detected. Upon composition analyses of individual subpopulations, LpA-I/A-II exhibited comparable proportions for total protein (∼58%), phospholipids (∼21%), total cholesterol (∼16%), triglycerides (∼5%), and free cholesterol (∼4%) across subfractions. LpA-I components, on the other hand, showed significant variability. This novel information about HDL subfractions will form a basis for an improved understanding of particle-specific functions of HDL.

Apolipoprotein A-II-mediated Conformational Changes of Apolipoprotein A-I in Discoidal High Density Lipoproteins

Background: Role of apolipoprotein (apo) A-II on metabolism of high density lipoproteins (HDLs) is unknown. Results: Conformational changes of apoA-I, the major apolipoprotein of HDL, caused by apoA-II in discoidal HDL are confined to two regions of apoA-I. Conclusion: Interactions between the two major apolipoproteins in discoidal HDL are site specific. Significance: Functional implications of HDL complexes will significantly benefit from such structural information. It is well accepted that HDL has the ability to reduce risks for several chronic diseases. To gain insights into the functional properties of HDL, it is critical to understand the HDL structure in detail. To understand interactions between the two major apolipoproteins (apos), apoA-I and apoA-II in HDL, we generated highly defined benchmark discoidal HDL particles. These particles were reconstituted using a physiologically relevant phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) incorporating two molecules of apoA-I and one homodimer of apoA-II per particle. We utilized two independent mass spectrometry techniques to study these particles. The techniques are both sensitive to protein conformation and interactions and are namely: 1) hydrogen deuterium exchange combined with mass spectrometry and 2) partial acetylation of lysine residues combined with MS. Comparison of mixed particles with apoA-I only particles of similar diameter revealed that the changes in apoA-I conformation in the presence of apoA-II are confined to apoA-I helices 3–4 and 7–9. We discuss these findings with respect to the relative reactivity of these two particle types toward a major plasma enzyme, lecithin:cholesterol acyltransferase responsible for the HDL maturation process.

Airway Peroxidases Catalyze Nitration of the β2-Agonist Salbutamol and Decrease Its Pharmacological Activity

β2-Agonists are the most effective bronchodilators for the rapid relief of asthma symptoms, but for unclear reasons, their effectiveness may be decreased during severe exacerbations. Because peroxidase activity and nitrogen oxides are increased in the asthmatic airway, we examined whether salbutamol, a clinically important β2-agonist, is subject to potentially inactivating nitration. When salbutamol was exposed to myeloperoxidase, eosinophil peroxidase or lactoperoxidase in the presence of hydrogen peroxide (H2O2) and nitrite (NO2−), both absorption spectroscopy and mass spectrometry indicated formation of a new metabolite with features expected for the nitrated drug. The new metabolites showed an absorption maximum at 410 nm and pKa of 6.6 of the phenolic hydroxyl group. In addition to nitrosalbutamol (m/z 285.14), a salbutamol-derived nitrophenol, formed by elimination of the formaldehyde group, was detected (m/z 255.13) by mass spectrometry. It is noteworthy that the latter metabolite was detected in exhaled breath condensates of asthma patients receiving salbutamol but not in unexposed control subjects, indicating the potential for β2-agonist nitration to occur in the inflamed airway in vivo. Salbutamol nitration was inhibited in vitro by ascorbate, thiocyanate, and the pharmacological agents methimazole and dapsone. The efficacy of inhibition depended on the nitrating system, with the lactoperoxidase/H2O2/NO2− being the most affected. Functionally, nitrated salbutamol showed decreased affinity for β2-adrenergic receptors and impaired cAMP synthesis in airway smooth muscle cells compared with the native drug. These results suggest that under inflammatory conditions associated with asthma, phenolic β2-agonists may be subject to peroxidase-catalyzed nitration that could potentially diminish their therapeutic efficacy.

Laser Desorption/Ionization (LDI)- and Maldi-Fourier Transform Ion Cyclotron Resonance Mass Spectrometric (FT/ICR/MS) Analysis of Hydrocarbon Samples

There are several distinct advantages to utilizing Fourier transform ion cyclotron resonance mass spectrometry (FT/ICR/MS) for the analysis of hydrocarbon materials. These advantages include the inherently high mass accuracy and resolution afforded by this technique (e.g., to distinguish between 12CH and 13C). In this chapter, we describe investigations of different sample preparation techniques for achieving high-resolution laser desorption/ionization (LDI)-FT/ICR/MS of metalloporphyrins, and we discuss the use of nonpolar matrices for the analysis of low molecular weight hydrocarbon materials.

Analysis of Polymeric Hydrocarbon Materials by Matrix-Assisted Laser Desorption/Ionization (Maldi) Mass Spectrometry

There are a number of analytical techniques available for the analysis of hydrocarbon polymeric materials. The most common and useful techniques include pyrolysis, chromatography, thermal analysis, spectroscopy (NMR, ER & Raman) and mass spectrometry.1 The growth of mass spectrometry for the analysis of synthetic polymers has nearly doubled in eight years (as measured by ASMS abstracts) as noted by the editor of the journal of the American Society for Mass Spectrometry (ASMS) in 1998.2 Among the many mass spectrometry techniques available, matrix-assisted laser desorption/ionization (MALDI) applications are constantly increasing. The MALDI technique was initially developed for biological molecules, mainly proteins and peptides,3,4 but was eventually utilized for other analytes including synthetic polymers 5. Although MALDI is quite successful for the analysis of polar polymers, it has not worked as well for nonpolar analytes such as hydrocarbon materials. This limitation is attributed to lack of proper understanding of some fundamental issues related to this technique such as the desorption/ionization mechanism and the role of the matrix in the MALDI process. In this chapter, MALDI-MS analytical approaches for the analysis of hydrocarbon polymeric materials are discussed.