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Separation of complex protein mixtures that have a wide dynamic range of concentration, such as plasma or serum, is a challenge for proteomic analysis. Sample preparation to remove high-abundant proteins is essential for proteomics analysis. Immunoglobulin yolk (IgY) antibodies have unique and advantageous features that enable specific protein removal to aid in the detection of low-abundant proteins and biomarker discovery. This report describes the efficiency and effectiveness of IgY microbeads in separating 12 abundant proteins from plasma with an immunoaffinity spin column or LC column. The protein separation and sample preparation process was monitored via SDS-PAGE, 2-DE, LC-MS/MS, or clinical protein assays. The data demonstrate the high specificity of the protein separation, with removal of 95-99.5% of the abundant proteins. IgY microbeads against human proteins can also selectively remove orthologous proteins of other mammals such as mouse, rat, etc. Besides the specificity and reproducibility of the IgY microbeads, the report discusses the factors that may cause potential variations in protein separation such as protein-protein interactions (known as "Interactome"), binding and washing conditions of immunoaffinity reagents, etc. A novel concept of Seppromics is introduced to address methodologies and science of protein separation in a context of proteomics.

Supplementation of vitamin E in NIDDM leads to enrichment of LDL and LDL subfractions and reduced susceptibility to oxidation. Despite a greater percentage increase in vitamin E content in small dense LDL, it remained substantially more susceptible to oxidation than was buoyant LDL. This suggests that dense, LDL may gain less protection against oxidation from antioxidant supplementation than does larger, more buoyant LDL. In contrast to previous reports, vitamin E supplementation did not reduce glycation of intracellular or plasma proteins.

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Background: Monitoring bone resorption with measurements of bone density and biochemical markers is indirect. We hypothesized that bone resorption can be studied directly by serial measurements of the ratio 41 Ca/Ca in serum after in vivo labeling of calcium pools with 41 Ca. We report the preparation of an intravenous 41 Ca dose suitable for humans, an analytical method for determining 41 Ca/Ca isotope ratios in biological samples, and studies in human volunteers. Methods:41 Ca was formulated and aliquoted into individual vials, and to the extent possible, the 41 Ca doses were tested according to US Pharmacopeia (USP) guidelines. A 10 nCi dose of 41 Ca was administered intravenously to 4 end stage renal disease (ESRD) patients on hemodialysis and 4 healthy control individuals. Distribution kinetics were determined over 168 days. Calcium was isolated with 3 precipitation steps and a cationexchange column, and 41 Ca/Ca ratios in serum were then measured by accelerator mass spectrometry. Results: The dosing solution was chemically and radiologically pure, contained <0.1 endotoxin unit/mL, and passed USP sterility tests. Quantification of 41 Ca/Ca ratios was linear from 6 ؋ 10 ؊14 to 9.1 ؋ 10 ؊10 . The run-to-run imprecision (as CV) of the method was 4% at 4.6 ؋ 10 ؊11 and 6% at 9.1 ؋ 10 ؊10 . The area under the curve of 41 Ca in the central compartment vs time was

Background: Microalbuminuria is an important prognostic marker in diabetic nephropathy and cardiovascular disease. Initially, most commercial assays used immunoreactivity to quantify microalbuminuria; however, size-exclusion HPLC demonstrated the existence of nonimmunoreactive forms of albumin that may not be detected by immunoassay. Recent liquid chromatography tandem mass spectrometry analyses suggested that size-exclusion HPLC gave higher results attributable to other urine proteins coeluting with albumin. We describe an assay that measures total microalbuminuria (immunoreactive and nonimmunoreactive) without any discernable interference from other common urine proteins. Methods: We used an automated chip electrophoresis system that utilized microfluidic separation technology and fluorescent sample detection. Each albumin specimen was mixed with the manufacturer's sample buffer in addition to a chicken albumin internal calibrator and then electrophoresed without additional reducing agents. Results: With variable concentrations of bovine serum albumin normalized to a chicken albumin internal calibrator, the electrophoresis system was best fit with a polynomial (R 2 ‫؍‬ 0.9997; concentration range, 5-300 mg/L). The lower limit of detection was 5 mg/L. Interchip and intrachip variation studies conducted on patient urine demonstrated CVs of 3%-13%. The introduction of potentially interfering agents (i.e., molecular analytes, nonalbumin proteins) did not alter precision. Compared with immunoassay, the chip electrophoresis identified higher microalbuminuria concentrations in

The application of gas chromatography/mass spectrometry (GC/MS) for the simultaneous quantitation of seven commonly encountered urinary benzodiazepine metabolites is described. After comparison of the signal-to-noise ratios of high mass ions of benzodiazepines using electron impact (EI), positive chemical ionization (PCI), and negative chemical ionization (NCI), NCI was chosen because of its increased sensitivity, which ranged from four to several thousand times that of either PCI or EI. This method is novel because NCI spectra for many of these compounds have not been described. For quantitation of benzodiazepines in urine, sample preparation consisted of enzymatic hydrolysis, liquid-liquid extraction, and reaction with a silylating reagent to form trimethylsilyl derivatives. The extraction efficiency of the method was greater than 70% (range, 73-89%) for nordiazepam, oxazepam, temazepam, lorazepam, N-1-hydroxyethylflurazepam, alpha-hydroxyal-prazolam, and alpha-hydroxytriazolam; the linear range for these compounds was from 50 to 2000 ng/mL. Within-run precision was less than 6% for all analytes in the range 50-2000 ng/mL; however, run-to-run precision ranged from 3 to 21%, depending on the analyte and concentration. Quantitation was based on area ratio of high mass ions relative to deuterated internal standards, acquired by scanning the mass range from m/z 250 to 450. Because these studies were performed in the scan mode, if desired, the sensitivity could be increased by using selected ion monitoring.