Apolipoprotein A-II-mediated conformational changes of apolipoprotein A-I in discoidal high density lipoproteins.

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 {beta}2-agonist salbutamol and decrease its pharmacological activity.

ß(2)-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 ß(2)-agonist, is subject to potentially inactivating nitration. When salbutamol was exposed to myeloperoxidase, eosinophil peroxidase or lactoperoxidase in the presence of hydrogen peroxide (H(2)O(2)) and nitrite (NO(2)(-)), 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 pK(a) 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 ß(2)-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/H(2)O(2)/NO(2)(-) being the most affected. Functionally, nitrated salbutamol showed decreased affinity for ß(2)-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 ß(2)-agonists may be subject to peroxidase-catalyzed nitration that could potentially diminish their therapeutic efficacy.

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.

Preterm labor biomarker discovery in serum using 3 proteomic profiling methodologies.

OBJECTIVE: The aim of this study was to identify changes in protein expression in normal pregnancy compared with preterm labor by using 3 proteomic methods. STUDY DESIGN: Serum was collected from 25 nonpregnant (n = 5) and pregnant women at 24-40 weeks' gestation (n = 20) who had preterm labor resulting in preterm delivery (n = 5), preterm labor with term delivery (n = 5), term labor resulting in delivery (n = 5), or at term with contractions (n = 5). Undepleted serum was used for surface-enhanced laser desorption ionization and immune-depleted serum for matrix-assisted laser desorption ionization and 2-dimensional electrophoresis. RESULTS: Surface-enhanced laser desorption ionization identified significantly different peaks between preterm labor resulting in preterm delivery vs term labor resulting in delivery and preterm labor resulting in preterm delivery vs preterm labor with term delivery using 4 surfaces. In preterm labor resulting in preterm delivery vs preterm labor with term delivery, a peak of 7783.2 m/z was significantly up-regulated and at 3164 m/z down-regulated on 3 surfaces. By using 2-dimensional electrophoresis, protein 5364 was significantly different between preterm labor resulting in preterm delivery and term labor resulting in delivery. In preterm labor resulting in preterm delivery, 6 proteins showed decreasing trend and 1 showed increasing trend vs preterm labor with term delivery. Matrix-assisted laser desorption ionization showed a striking difference at 55,000 m/z between preterm labor resulting in preterm delivery and term labor resulting in delivery. CONCLUSION: Surface-enhanced laser desorption ionization identified 2 proteins fulfilling the criteria of putative biomarkers. Biomarker identification may aid in identifying women with preterm labor who will deliver preterm.

Mass spectrometric determination of apolipoprotein molecular stoichiometry in reconstituted high density lipoprotein particles.

Plasma HDL-cholesterol and apolipoprotein A-I (apoA-I) levels are strongly inversely associated with cardiovascular disease. However, the structure and protein composition of HDL particles is complex, as native and synthetic discoidal and spherical HDL particles can have from two to five apoA-I molecules per particle. To fully understand structure-function relationships of HDL, a method is required that is capable of directly determining the number of apolipoprotein molecules in heterogeneous HDL particles. Chemical cross-linking followed by SDS polyacrylamide gradient gel electrophoresis has been previously used to determine apolipoprotein stoichiometry in HDL particles. However, this method yields ambiguous results due to effects of cross-linking on protein conformation and, subsequently, its migration pattern on the gel. Here, we describe a new method based on cross-linking chemistry followed by MALDI mass spectrometry that determines the absolute mass of the cross-linked complex, thereby correctly determining the number of apolipoprotein molecules in a given HDL particle. Using well-defined, homogeneous, reconstituted apoA-I-containing HDL, apoA-IV-containing HDL, as well as apoA-I/apoA-II-containing HDL, we have validated this method. The method has the capability to determine the molecular ratio and molecular composition of apolipoprotein molecules in complex reconstituted HDL particles.

Investigation of single-site zirconium azaborolinyl complexes by laser desorption ionization time of flight mass spectrometry.

Laser desorption ionization mass spectrometry (LDI-MS) was performed on selected azaborolinyl zirconium complexes in order to study them as potential industrial polyolefin catalyst compounds. UV-vis absorption data in conjunction with negative and positive ionization analyses revealed the mechanistic workings of the compounds when such single-site catalysts function in olefin polymerization reactions. Results presented highlight the many benefits of LDI-MS in the study of single-site zirconium azaborolinyl catalyst complexes including minimal sample preparation, the absence of matrix/pH/solvation effects, a lower degree of fragmentation as compared to EI ionization, high sensitivity and the ability to observe interesting and unique gas phase chemistry including ring slippage, and distinctive metal coordination and oxidation states. The extreme lability of the chlorine ligands was apparent, as well as the reactivity of the resulting zirconium metal center as demonstrated by the appearance of numerous similar four-coordinate zirconium species along with di-chloro-bridged dimers. LDI-MS permits analysis at low molecular weights, improving spectral characterization of these zirconium complexes.

Peroxynitrite treatment in vitro disables catalytic activity of recombinant p38 MAPK.

Protein tyrosine nitration is a post-translational modification occurring under conditions of oxidative stress in a number of diseases. The causative agent of tyrosine nitration is the potent prooxidant peroxynitrite that results from the interaction of nitric oxide and superoxide. We have previously demonstrated existence of nitrotyrosine in placenta from pregnancies complicated by preeclampsia, which suggested the possibility of the existence of nitrated proteins. Nitration of various proteins has been demonstrated to more commonly result in loss of protein function. Potential nitration of p38 MAPK, a critical signaling molecule has been suggested and also tentatively identified in certain in vivo systems. In this study we demonstrate for the first time nitration of recombinant p38 MAPK in vitro and an associated loss of its catalytic activity. LC-MS data identified tyrosine residues Y132, Y245 and Y258 to be nitrated. Nitration of these specific residues was deduced from the 45.0-Da change in mass that these residues exhibited that was consistent with the loss of a proton and addition of the nitro group.

A three-dimensional molecular model of lipid-free apolipoprotein A-I determined by cross-linking/mass spectrometry and sequence threading.

Apolipoprotein (apo) A-I, a 243-residue, 28.1-kDa protein is a major mediator of the reverse cholesterol transport (RCT) pathway, a process that may reduce the risk of cardiovascular disease in humans. In plasma, a small fraction of lipid-free or lipid-poor apoA-I is likely a key player in the first step of RCT. Therefore, a basic understanding of the structural details of lipid-free apoA-I will be useful for elucidating the molecular details of the pathway. To address this issue, we applied the combined approach of cross-linking chemistry and high-resolution mass spectrometry (MS) to obtain distance constraints within the protein structure. The 21 lysine residues within apoA-I were treated with homo bifunctional chemical cross-linkers capable of covalently bridging two lysine residues residing within a defined spacer arm length. After trypsin digestion of the sample, individual peptide masses were identified by MS just after liquid chromatographic separation. With respect to the linear amino acid sequence, we identified 5 short-range and 12 long-range cross-links within the monomeric form of lipid-free apoA-I. Using the cross-linker spacer arm length as a constraint for identified Lys pairs, a molecular model was built for the lipid-free apoA-I monomer based on homology with proteins of similar sequence and known three-dimensional structures. The result is the first detailed model of lipid-free apoA-I. It depicts a helical bundle structure in which the N- and C-termini are in close proximity. Furthermore, our data suggest that the self-association of lipid-free apoA-I occurs via C- and N-termini of the protein based on the locations of six cross-links that are unique to the cross-linked dimeric form of apoA-I.