Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple possible functions that are likely influenced by glycosylation. Identification of oligosaccharide attachment sites and structural characterization of oligosaccharides in human serum clusterin has been performed by mass spectrometry and Edman degradation. Matrix-assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holoclusterin (58,505 f 250 and 63,507 * 200). Mass spectrometry also revealed molecular heterogeneity associated with both the LY and p subunits of clusterin, consistent with the presence of multiple glycoforms. The data indicate that clusterin contains 17-27% carbohydrate by weight, the LY subunit contains 0-30% carbohydrate and the p subunit contains 27-30% carbohydrate.Liquid chromatography electrospray mass spectrometry with stepped collision energy scanning was used to selectively identify and preparatively fractionate tryptic glycopeptides. Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites within each peptide. A total of six N-linked glycosylation sites were identified, three in the LY subunit ( d 4 N , cy*lN, a I z 3 N ) and three in the p subunit (p@N, p'27N, and @'47N). Seven different possible types of oligosaccharide structures were identified by mass including: a monosialobiantennary structure, bisialobiantennary structures without or with one fucose, trisialotriantennary structures without or with one fucose, and possibly a trisialotriantennary structure with two fucose and/or a tetrasialotriantennary structure. Site p@N exhibited the least glycosylation diversity, with two detected types of oligosaccharides, and site pI4'N exhibited the greatest diversity, with five or six detected types of oligosaccharides. Overall, the most abundant glycoforms detected were bisialobiantennary without fucose and the least abundant were monosialobiantennary, trisialotriantennaq with two fucose and/or tetrasialotriantennary. Clusterin peptides accounting for 99% of the primary structure were identified from analysis of the isolated LY and p subunits, including all Ser-and Thr-containing peptides. No evidence was found for the presence of 0-linked or sulfated oligosaccharides. The results provide a molecular basis for developing a better understanding of clusterin structure-function relationships and the role clusterin glycosylation plays in physiological function.

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Muller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 1 1-cis-retinal and/or 1 1-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein.Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 1 5 2 0 % of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state I9F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies.

The cellular retinaldehyde-binding protein (CRALBP) 1 is thought to play a fundamental role in vitamin A metabolism in the retina and retinal pigment epithelium (RPE). Notably, mutations in the human CRALBP gene can result in autosomal recessive retinitis pigmentosa (1). In vitro CRALBP serves as a substrate carrier protein for enzymes of the mammalian visual cycle, modulating whether 11-cis-retinol (11-cis-Rol) is stored as an ester in the RPE or oxidized by 11-cis-Rol dehydrogenase to 11-cis-retinal (11-cis-Ral) for visual pigment regeneration (2). In the RPE and Mü ller cells of the retina, CRALBP carries endogenous 11-cis-retinoids, the isomers of vitamin A utilized for phototransduction. However, CRALBP is not always associated with a retinoid ligand and more than one physiological role for the protein appears likely (3). The protein is also present in ciliary body, cornea, pineal gland, optic nerve, brain, transiently in iris, but not in the rod and cone photoreceptors. CRALBP is expressed in developing retina and RPE before the tissues contain 11-cis-retinoids or the enzyme responsible for generating 11-cis-retinoids (3). Apparently the protein serves functions unrelated to visual pigment regeneration in brain and tissues not involved in the visual cycle and may bind ligands other than retinoids.CRALBP was first detected in retina about 20 years ago and shown to carry 11-cis-Rol and 11-cis-Ral as endogenous ligands (4,5). Structure function studies have defined ligand stereoselectivity and photosensitivity (6), developed a topological and epitope map (7), established in vitro evidence for a substrate carrier function in RPE (8, 9) and produced human recombi-

An automated chip-based infusion nanoelectrospray ionization (nanoESI) platform was used to demonstrate reproducible quantitation of drug molecules from biological matrices. Three sample preparation strategies were explored including protein precipitation of plasma with acetonitrile, de-salting of the plasma, and a combination of protein precipitation with subsequent de-salting of the dried and reconstituted extract. The best results were obtained when fortified human plasma samples containing midazolam were precipitated with acetonitrile containing alprazolam as the internal standard (IS). The supernatant was concentrated to dryness, reconstituted in aqueous acid, and de-salted by automated reversed-phase solid-phase extraction (SPE) prior to infusion nanoESI-MS/MS. Analyses employed a triple quadrupole mass spectrometer operated in selected reaction monitoring (SRM) mode. Each sample was infused for approximately 10 s and the resulting ion current profiles were integrated. Area ratios were used for regression analysis of standard samples (1.5-500 ng/mL). Quality control samples (3, 250, and 400 ng/mL) in five replicates from three different analysis days demonstrated intra-assay precision (< or =16%), inter-assay precision (< or =5%), and overall accuracy (+/-9% deviation). Infusion reproducibility of the assay was established by analyzing extracts after storage for 24 h at ambient temperature. Control plasma samples from six different sources probed the potential utility of this technique for the analysis of clinical samples. At the lower limit of quantitation (LLQ), variability and mean overall accuracy were < or =13% CV and +/-3% deviation, respectively, while at the upper limit of quantitation (ULQ) variability and mean overall accuracy were < or =9% CV and +/-9% deviation, respectively. Inter-chip variability was established by determining standard sample extracts across five different chips (< or =12% CV). Throughput for the assay was 55 s per sample, although this time may be shortened to 40 s per sample with recent improvements in the automated nanoESI system. No contamination or carryover was observed using this promising automated nanoESI-MS/MS platform.

A liquid chromatography/high-field asymmetric waveform ion mobility spectrometry/tandem mass spectrometry (LC-FAIMS-MS/MS) semi-quantitative method was developed for the simultaneous determination of prostaglandin (PG) E2, PGD2, PGF(2alpha), 6-keto-PGF(1alpha), and thromboxane (TX) B2. Diluted samples containing these prostanoids and their tetra-deuterium-substituted internal standards were analyzed by LC followed by either selected reaction monitoring (LC-SRM) or FAIMS and selected reaction monitoring (LC-FRM). FAIMS reduced background noise, separated the isobaric ions PGE2 and PGD2, and separated dynamically interchanging TXB2 anomers. This is the first report of the separation of interconverting anomers by FAIMS. Generally, the LC-FRM chromatograms were more selective than the LC-SRM chromatograms. Its potential was demonstrated by analysis of prostanoids in guinea pig lumbar spinal cord homogenate.

The effect of metabolite interference during liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of an amine drug was investigated using FAIMS (high-Field Asymmetric waveform Ion Mobility Spectrometry). The selected reaction monitoring (SRM) transition used for the drug exhibited an interference due to in-source conversion of the N-oxide metabolite to generate an ion isobaric with the drug. The on-line FAIMS device removed the metabolite interference before entrance to the mass spectrometer. FAIMS was used to demonstrate the relative accuracy and precision of drug analysis even in the presence of a co-eluting metabolite that may undergo insource conversion. Copyright # 2005 John Wiley & Sons, Ltd.Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) is a standard technique for bioanalysis in the pharmaceutical industry.1-4 One of the main reasons for the success of LC/MS/MS is the very high level of selectivity that can be routinely achieved. This selectivity is due to the distinct nature of the separation mechanisms of each individual technique involved in LC/MS/MS bioanalysis. The condensed-phase LC separation provides selectivity based on the physicochemical properties of the analyte in relation to mobile and stationary phases. In contrast, the gas-phase MS/MS separation offers selectivity based on mass-to-charge (m/z) ratio differences as well as chemical fragmentation. When these two orthogonal techniques are used together, the combined selectivity allows for very low limits of quantitation with accurate determinations of drug levels in complex matrices. In addition to enhanced selectivity, the introduction of LC/ MS/MS has demonstrated the potential for increases in sample throughput. In the race to produce the next blockbuster drug, pharmaceutical companies and contract research organizations analyze many samples in increasingly shorter periods of time. Common bioanalytical laboratory practices to reduce this time-to-market include minimizing the effort put into sample preparation, reducing or eliminating chromatographic analyses, and using elaborate MS n techniques.5-9 However, the race for higher throughput must be balanced with the requirement of maintaining sufficient selectivity. One potential source for loss of selectivity in LC/ MS/MS bioanalysis is the interference of a metabolite that can undergo in-source conversion. The interference problem arises from the fact that the metabolite can produce a molecular ion identical to the molecular ion of the drug. This conversion may be due to thermal decomposition during sample ionization, which has been observed during atmospheric pressure chemical ionization (APCI). 10 In contrast, the conversion may be the result of the cone voltage causing in-source collisionally activated dissociation (in-source CAD), as ions enter the moderate-pressure region of the vacuum chamber prior to mass analysis. The presence of a prodrug, due to incomplete in vivo conversion into the intended drug, could also cause a similar interference in t...

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