Variable fragmentation and ionization of amyloid-beta epimers and isomers.

While the existence of D-amino acids in peptides and proteins has recently been accepted in higher forms of life, their roles and importance are yet to be understood. The lack of analytical methods present for such epimeric and/or isomeric analyses often limits developments in the field. Studies have shown the elevated presence of epimeric and isomeric modifications to amyloid-beta (Aß) peptides extracted from Alzheimer's disease patients. These modifications most frequently occur through aspartic acid and serine residues. Because such peptides are indistinguishable by mass alone, selective liquid chromatography tandem mass spectrometry analysis is required to differentiate such peptides. Herein, we examine MS/MS of tryptic fragments of Aß peptides containing D-Asp, L-iso-Asp, D-iso-Asp, and/or D-Ser modifications. Peptide ionizability and fragmentation are explored through selected reaction monitoring, selected ion monitoring, and product ion scan. The results show the variability of ionization and fragmentation for many "identical mass peptides" and how these differences can affect the analysis of isomeric and epimeric peptides.

Antibody binding of amyloid beta peptide epimers/isomers and ramifications for immunotherapies and drug development.

Extracellular deposition of amyloid beta (Aß) peptide is a contributing factor of Alzheimer's disease (AD). Considerable effort has been expended to create effective antibodies, or immunotherapies, targeting Aß peptides. A few immunotherapies are thought to provide some benefit. It is possible that a contributing factor to the responses of such therapies may be the presence of modified, or aberrant, Aß peptides found in AD patients. These aberrations include the isomerization and epimerization of L-Asp and L-Ser residues to form D-Asp, L/D-isoAsp, and D-Ser residues, respectively. An effective methodology is essential to isolate all Aß peptides and then to quantify and locate the aberrant amino acids. Modifications to Aß peptides may elevate the deposition of Aß plaques and/or contribute to the neurodegeneration in AD patients, and may alter the binding affinity to antibodies. Herein, we used immunoprecipitation to examine the binding affinity of four antibodies against 18 epimeric and/or isomeric Aß peptides compared to wild type (all L) Aß peptide. Tandem mass spectrometry was used as a detection method, which also was found to produce highly variable results for epimeric and/or isomeric Aß.

Comprehensive chiral GC-MS/MS and LC-MS/MS methods for identification and determination of N-acyl homoserine lactones.

N-acyl homoserine lactones (N-HLs) are signaling molecules synthesized by gram-negative bacteria to communicate in a process called quorum sensing. Most reported methods for the analysis of N-HLs, which are chiral molecules, do not distinguish between enantiomers. Typical examples include biosensors, liquid chromatography with UV detection, gas chromatography coupled with a mass spectrometer (GC-MS) and liquid chromatography coupled with mass spectrometer (LC-MS). Recently, the production of both D,L-N-HLs have been reported in Vibrio fischeri and Burkholderia cepacia. Concentrations of the D-N-HLs were found at the limit of quantification for the employed method. Therefore, for further studies of the role of the D-N-HLs in bacterial physiology, more sensitive, reliable, and selective analytical methods are necessary. In this work, such comprehensive chiral analytical methods for the identification and determination of 18 N-HLs using solid phase extraction followed by GC-MS/MS and LC-MS/MS analyses were developed. Extraction recoveries for the more hydrophilic C4 N-HLs were <10% of all other N-HLs, thus offering a possible explanation as to their lack of detection in previous studies. The chiral separations of all 18 N-HLs derivatives were accomplished by the complementary GC-MS/MS and LC-MS/MS methods. The limit of detection for LC-MS/MS method was as low as 1 ppb. The limit of detection for the GC-MS/MS method was found to be one to three orders of magnitude higher than the LC-MS/MS method. Due to the high extraction recovery and a preconcentration factor of 100, concentrations as low as 10 ppt can be detected by LC-MS/MS in biological samples. The LC-MS/MS approach provided greater enantioselectivity for the larger, more hydrophobic N-HLs while GC-MS/MS provided better enantioselectivity for the smaller N-HLs.

Production of both l- and d- N-acyl-homoserine lactones by Burkholderia cepacia and Vibrio fischeri.

Quorum sensing (QS) is a complex process in which molecules, such as l-N-acyl-homoserine lactones (l-AHLs), are produced as essential signaling molecules allowing bacteria to detect and respond to cell population density by gene regulation. Few studies have considered the natural production and role of the opposite enantiomers, d-AHLs. In this work, production of d,l-AHLs by Burkholderia cepacia and Vibrio fischeri was monitored over time, with significant amounts of d-AHLs detected. Bioluminescence of V. fischeri was observed with maximum bioluminescence correlating with the maximum concentrations of both l- and d- octanoyl-homoserine lactones (l- and d-OHL). l-Methionine, a precursor to l-AHLs, was examined via supplementation studies conducted by growing three parallel cultures of B. cepacia in M9 minimal media with added l-, d-, or d,l-methionine and observing their effect on the production of d,l-AHL by B. cepacia. The results show that addition of any methionine (l-, d-, or d,l-) does not affect the overall ratio of l- to d-AHLs, that is d-AHL production was not selectively enhanced by d-methionine addition. However, the overall AHL (l- and d-) concentration does increase with the addition of any methionine supplement. These findings indicate the possibility of a distinct biosynthetic pathway for d-AHL production, possibly exposing a new dimension within bacterial communication.

Rapid and selective separation of amyloid beta from its stereoisomeric point mutations implicated in neurodegenerative Alzheimer's disease.

Extracellular deposition of amyloid beta (Aß) peptides are a hallmark of Alzheimer's disease. The isomerization and epimerization of Aß peptides have been linked to the enhanced deposition of Aß plaques. Therefore, considerable effort has been expended to create effective methods to distinguish such aberrant Aß peptides from normal Aß peptides. Herein, we have developed chromatographic retention U-shaped curves to investigate the hydrophobicity of Aß 1-38, 1-40, 1-42 and fourteen aberrant Aß 1-42 peptides. Using this information, we developed the first selective and comprehensive method that can easily detect both aberrant and normal Aß peptides simultaneously using high performance liquid chromatography-mass spectrometry (HPLC-MS). We show for the first time that D-Ser modifications to Aß cause the peptide to be more hydrophilic, as does D-Asp and L/D-iso-Asp.

Enantiomeric separation of quorum sensing autoinducer homoserine lactones using GC-MS and LC-MS.

Homoserine lactones (HSLs) are signaling molecules synthesized by Gram-negative bacteria in order to communicate in a process termed "quorum sensing." Until recently, only the L-stereoisomers of HSLs were thought to be produced and able to incite quorum sensing. However, recent studies have shown that select Gram-negative bacteria additionally produce non-trivial amounts of D-HSLs which may also play a role in quorum sensing. Current methods for the separation of HSL enantiomers cannot effectively separate all classes of HSLs and its enantiomers. More robust methods of separation and detection of D-HSLs are necessary. We have developed rapid and selective methods using liquid chromatography (LC) and gas chromatography (GC) coupled with mass spectrometry (MS) which can simultaneously enantiomerically separate all classes of HSLs. The advantages of these methods are in the MS compatibility as well as the ability to enantiomerically separate all classes of HSLs in a single run. The first enantiomeric separations of oxo- and hydroxy-HSLs by GC-MS, through the use of N,O-bis(trimethylsilyl)trifluoroacetamide-derivatizing reagents are discussed. Graphical Abstract.

Complete identification of all 20 relevant epimeric peptides in ß-amyloid: a new HPLC-MS based analytical strategy for Alzheimer's research.

Although the underlying cause of Alzheimer's disease (AD) is not known, the extracellular deposition of ß-amyloid (Aß) is considered as a hallmark of AD brains. Evidence has shown the occurrence of d-Asp, isoAsp, and d-Ser residues in Aß, which may be indicative of and/or contribute to the neurodegeneration in AD patients. Herein, we have developed the first high-throughput profiling technique for all 20 isobaric Aß peptide epimers containing Asp, isoAsp, and Ser isomers using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). This new analytical strategy allows the direct detection and identification of all possible Asp, isoAsp, and Ser stereoisomers in Aß, and may contribute to a better understanding of the pathogenesis of AD.

Enantiomeric impurities in chiral catalysts, auxiliaries, and synthons used in enantioselective syntheses. Part 5.

The enantiomeric excess of chiral starting materials is one of the important factors determining the enantiopurity of products in asymmetric synthesis. Fifty-one commercially available chiral reagents used as building blocks, catalysts, and auxiliaries in various enantioselective syntheses were assayed for their enantiomeric purity. The test results were classified within five impurities level (ie, <0.01%, 0.01%-0.1%, 0.1%-1%, 1%-10%, >10%). Previously from 1998 to 2013, several reports have been published on the enantiomeric composition of more than 300 chiral reagents. This series of papers is necessitated by the fact that new reagents are forthcoming and that the enantiomeric purity of the same reagent can vary from batch to batch and/or from supplier to supplier. This report presents chiral liquid chromatography (LC) and gas chromatography (GC) methods to separate enantiomers of chiral compounds and evaluate their enantiomeric purities. The accurate and efficient LC analysis was done using newly introduced superficially porous particle (SPP 2.7 µm) based chiral stationary phases (TeicoShell, VancoShell, LarihcShell-P, and NicoShell).