A Cluster of Proteins Implicated in Kidney Disease Is Increased in High-Density Lipoprotein Isolated from Hemodialysis Subjects.

Cardiovascular disease is the leading cause of death in end-stage renal disease (ESRD) patients treated with hemodialysis. An important contributor might be a decline in the cardioprotective effects of high-density lipoprotein (HDL). One important factor affecting HDL's cardioprotective properties may involve the alterations of protein composition in HDL. In the current study, we used complementary proteomics approaches to detect and quantify relative levels of proteins in HDL isolated from control and ESRD subjects. Shotgun proteomics analysis of HDL isolated from 20 control and 40 ESRD subjects identified 63 proteins in HDL. Targeted quantitative proteomics by isotope-dilution selective reaction monitoring revealed that 22 proteins were significantly enriched and 6 proteins were significantly decreased in ESRD patients. Strikingly, six proteins implicated in renal disease, including B2M, CST3, and PTGDS, were markedly increased in HDL of uremic subjects. Moreover, several of these proteins (SAA1, apoC-III, PON1, etc.) have been associated with atherosclerosis. Our observations indicate that the HDL proteome is extensively remodeled in uremic subjects. Alterations of the protein cargo of HDL might impact HDL's proposed cardioprotective properties. Quantifying proteins in HDL may be useful in the assessment of cardiovascular risk in patients with ESRD and in assessing response to therapeutic interventions.

Humans with atherosclerosis have impaired ABCA1 cholesterol efflux and enhanced high-density lipoprotein oxidation by myeloperoxidase.

RATIONALE: The efflux capacity of high-density lipoprotein (HDL) with cultured macrophages associates strongly and negatively with coronary artery disease status, indicating that impaired sterol efflux capacity might be a marker-and perhaps mediator-of atherosclerotic burden. However, the mechanisms that contribute to impaired sterol efflux capacity remain poorly understood. OBJECTIVE: Our aim was to determine the relationship between myeloperoxidase-mediated oxidative damage to apolipoprotein A-I, the major HDL protein, and the ability of HDL to remove cellular cholesterol by the ATP-binding cassette transporter A1 (ABCA1) pathway. METHODS AND RESULTS: We quantified both site-specific oxidation of apolipoprotein A-I and HDL's ABCA1 cholesterol efflux capacity in control subjects and subjects with stable coronary artery disease or acute coronary syndrome. Subjects with coronary artery disease and acute coronary syndrome had higher levels of chlorinated tyrosine 192 and oxidized methionine 148 compared with control subjects. In contrast, plasma levels of myeloperoxidase did not differ between the groups. HDL from the subjects with coronary artery disease and acute coronary syndrome was less able to accept cholesterol from cells expressing ABCA1 compared with HDL from control subjects. Levels of chlorinated tyrosine and oxidized methionine associated inversely with ABCA1 efflux capacity and positively with atherosclerotic disease status. These differences remained significant after adjusting for HDL-cholesterol levels. CONCLUSIONS: Our observations indicate that myeloperoxidase may contribute to the generation of dysfunctional HDL with impaired ABCA1 efflux capacity in humans with atherosclerosis. Quantification of chlorotyrosine and oxidized methionine in circulating HDL might be useful indicators of the risk of cardiovascular disease that are independent of HDL-cholesterol.

Preparative separation of a multicomponent peptide mixture by mass spectrometry.

We report on the first multiplex preparative separation by mass spectrometry of bio-organic molecules in the 200-350 Da mass range that is typical for synthetic drugs. A five-component mixture consisting of two di- and three tripeptides has been separated by mass using a specially designed mass spectrometer. The instrument for preparative separations consists of an electrospray ionization (ESI) source, ion transfer optics, an electrostatic sector, and an inhomogeneous-field magnetic mass analyzer that achieves linear mass dispersion of ion beams. Protonated peptides produced by electrospray were separated, nondestructively landed on a 16-channel array of dry collector plates, and reconstituted in solution. The preparation procedures and the instrumental conditions have been optimized to maximize the ion currents. The significant features of the special mass spectrometer are high ion currents and simultaneous separation and collection of mixture components.

Preparative separation of mixtures by mass spectrometry.

A specially designed mass spectrometer which allows for preparative separation of mixtures is described. This mass spectrometer allows for large ion currents, on the order of nanoamperes, to be produced by electrospray and transmitted into a high vacuum. Accumulation of nanomole quantities of collected and recovered material in several hours is demonstrated. The use of high-velocity ions reduces space charge effects at high ion currents. Separation of mass occurs simultaneously for all ions, providing a 100% duty cycle. The use of a linear dispersion magnet avoids compression at higher m/z ratios. A deceleration lens slows the ions to allow for soft landing at low kinetic energy. The ions are neutralized by ion pairing on an oxidized metal surface. Retractable landing plates allow for easy removal of the separated components.

Gas-phase halonium metathesis and its competitors. Skeletal rearrangements of cationic adducts of saturated ketones.

The methyl cation and CF(3)(+) attack saturated, acyclic ketones to make vibrationally excited adduct ions. Despite their high internal energies and short lifetimes, these adducts undergo deep-seated rearrangements that parallel slower processes in solution. Observed pathways include alkene and alkane expulsions, in addition to (in the case of CF(3)(+)) the precedented loss of CF(2)O + HF. For the vast majority of ketones, the principal charged products are the CF(3)(+) adducts of lighter carbonyl compounds, ions that are not easily prepared by other avenues. Evidence for ion structures comes from collisionally activated unimolecular decomposition and bimolecular ion-molecule reactions. Typical examples are di-n-propyl and diisopropyl ketones (both of which produce CH(3)CH=OCF(3)(+) as the principal ion-molecule reaction product) and pentamethylacetone (which produces (CH(3))(2)C=OCF(3)(+) as virtually the sole ion-molecule reaction product). Isotopic labeling experiments account for mechanisms, and DFT calculations provide a qualitative explanation for the relative abundances of products from unimolecular decompositions of the chemically activated CF(3)(+) adduct ions that are initially formed.

Gas-phase electrophilic attack of a double bond exhibits stereoselectivity.

Protonated acetaldehyde (ion 1) reacts with allyltrimethylsilane (allyl-TMS) in the gas phase to yield cis-piperylene (cis-1,3-pentadiene) as the major product. The cis isomer predominates over trans by a factor >/=15:1, a degree of stereoselectivity that is unprecedented in a reaction where the double bond geometry has not been specified in the reactant. The neutral products were assessed by creating tritiated 1 via decay of a tritium nucleus in gaseous ethanol molecules labeled with >1 tritium atom. The radioactive C5H8 products must result from addition of the electrophilic ion to the allyl group followed by an elimination. Deprotonation of C5H9+ cannot account for the product stereochemistry. One possible explanation is that addition of the electrophile to the double bond is followed by elimination of Me3SiOH2+ on a time scale faster than that by which the initially formed adduct ion can change its conformation.