Abstract An analytical method has been developed that can provide more reliable analytical information than the classical fractionation method which has been used by soil scientists for years to determine heavy metal contents of soil, sediment, and sludge samples. The new approach utilizes d.c. plasma atomic emission spectrometry in combination with ion chromatography (DCPAES-IC), whereby the DCPAES provides element selective measurements of the chromatographic effluents. In this way, the combined analytical system provides information on all the species of a metal present in the different steps of the fractionation approach. The DCPAES-IC approach also addresses questions pertaining to completeness of extraction, interference, and reagent concentration effects.
In this study, polyaromatic hydrocarbon compounds including anthracene, pyrene, acenaphthene and perylene were investigated as nonpolar matrices in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of selected nonpolar analytes. First, the influence of matrix and the analyte ionization potentials on a charge-transfer ionization mechanism were determined. Results of these studies demonstrated that formation of radical molecular cations in MALDI-MS depends on the ionization energy difference between the matrix and the analyte. Charge-transfer ionization occurs only when the ionization potential of the matrix is higher than that of the analyte. Next, nonpolar matrices were investigated for the analysis of low molecular weight nonpolar polymers including polybutadiene, polyisoprene, and polystyrene. Conventionally, these types of polymers are analyzed by MALDI-MS using acidic matrices, such as all-trans-retinoic acid, with an additional metal salt as a cationization reagent. Nonpolar matrices were shown to be more effective than acidic matrices, as nonpolar matrices provide better matrix isolation and enhanced spectra reproducibility. Potential difficulties associated with background silver salt clusters present during the analysis of nonpolar polymers by MALDI-MS are reported. Silver cluster ions are observed with m/z values ranging from 1500 to about 7000 when acidic, polar matrices are used with silver salts. It was found that these background signals could be greatly reduced by the use of the nonpolar matrices. Alternatively, using copper salts with acidic matrices instead of silver salts substantially improves the mass spectral quality due to reduced background signals. Finally, the nonpolar matrices were applied to other polymeric compounds including amine functionalized polystyrene, platinum (II) linked modified polystyrenes, and poly[(o-cresyl glycidyl ether)- co-formaldehyde] (PGF). These studies demonstrated that nonpolar matrices can be used for the MALDI-MS analysis of polymers of varying polarity. As with the prior studies, nonpolar matrices were found to be generally more effective for the analysis of these analytes than acidic matrices. Taken together, these studies demonstrate that nonpolar matrices provide high quality MALDI mass spectra of nonpolar and moderately nonpolar polymers. These improvements arise due to improved matrix-analyte miscibility as well as reduction in background salt clusters when silver salts are used as cationization reagents.
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.
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.
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 5,000. The standard MALDI-MS approach for the analysis of these types of polymers involves the use of conventional acidic matrices, such as all-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.
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.
