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Abstract A traditional immobilized pH gradient (IPG) has a high stability for isoelectric focusing (IEF) but suffers from time-consuming rehydration, focusing and staining-imaging as well as complex performance. To address these issues, an IEF system with an array of 24 IPG columns (10 mm × 600 μm × 50 μm) and dynamic scanning imaging (DSI) was firstly designed for protein focusing. Moreover, two IPG columns (pH 4–9 and pH 6.7–7.7 of 10 mm in length) were firstly synthesized for IEF. A series of experiments were carried out based on the IEF array. In contrast to a traditional IPG IEF with more than 10 h rehydration, 5–14 h IEF and ca 10 h stain-imaging, the IEF array had the following merits: 25 min rehydration for sample loading, 4 min IEF, and 2 min dynamics scanning of 24 columns, well addressing the issues of traditional IEF. Furthermore, the IEF array had fair sensitivity (LOD of 60 ng), good recovery (95%), and high stability (1.02% RSD for intra-day and 2.16% for inter-day). Finally, the developed array was successfully used for separation and determination of HbA 1c (a key biomarker for diabetes diagnosis) in blood samples. All these results indicated the applicability of the developed IEF array to diabetes diagnosis. Graphical abstract Image 1 Highlights • A novel IEF system with an array of 24 IPG columns and dynamic scanning imaging (DSI). • Merits of fast rehydration of 25 min, quick focusing of 4 min and 2 min DSI for 24 IPG column assay. • First report on efficient quantitation of HbA 1c in diabetes sample via IEF array but not HPLC. [ABSTRACT FROM AUTHOR]

Circulating tumor exosomes harbor plenty of cancer biological information, which have emerged as promising targets for cancer early detection and diagnosis. Human serum and saliva are unique diagnostic body fluids, which contain numerous circulating exosomes. It is necessary to establish standardized isolation method and compare their proteome profiling for translational medicine. High abundant proteins in these body fluids were removed before exosomes isolation and obtained exosomes were further confirmed by morphology analysis and surface biomarker test. Label free quantification was applied to systematically compare the protein profiling in saliva and serum exosomes. 319 and 994 exosomal proteins were identified from saliva and serum by LC-MS/MS, respectively. To explore their utility for cancer proteomics, we systematically compared the proteome of saliva and serum exosomes from healthy subjects and lung cancer patients. In particular, 11 potential candidates were coincidently discovered in both body fluids of lung cancer patients. Our finding enforced the hypothesis that cancer related proteins were presented in saliva and serum exosomes, which promoted the unique features of exosomes in our body fluids. A circulating exosomes based body fluid test could be easily established for monitoring cancer once these candidates were validated. [ABSTRACT FROM AUTHOR]

Mitochondria play essential roles in both energy metabolism and cell signaling, which are critical for cell survival. Although significant efforts have been invested in understanding mitochondrial biology, methods for intact mitochondria preparation are technically challenging and remain to be improved. New methods for heterogeneous mitochondria purification will therefore boost our understanding on their physiological and biophysical properties. Herein, we developed a novel recycling free-flow isoelectric focusing (RFFIEF) with post-pH gradient sample injection (post-PGSI) for preparative separation of mitochondria. Crude mitochondria of rabbit liver obtained from differential centrifugation were purified by the developed method according to their pI values as six fractions. Transmission electron microscope images revealed that intact mitochondria existed in two fractions of pH 6.24 and 6.61, degenerative mitochondria were in two fractions of pH 5.46 and 5.72, and inner membrane vesicles (IMVs) appeared in the fractions of pH 4.70 and 5.04. Membrane potential measurement proved a dramatic difference between intact mitochondria and IMVs, which reflected the bioactivity of obtained populations. Particularly, proteomics analyses revealed that more number of proteins were identified in the intact fractions than that of IMVs or crude mitochondria, which demonstrated that RFFIEF could be powerful tool for the preparation of intact organelle as well as their proteomic and in-depth biological analysis. [ABSTRACT FROM AUTHOR]

Highlights: [•] A novel moving reaction boundary model formed with base and immobilized protein. [•] The moving reaction boundary titration developed for protein content assay. [•] Determining protein content via visual boundary displacement signal. [•] Weak influence of non-protein nitrogen reagents on the developed method. [•] Fast and sensitive analytical method as compared with classic Kjeldahl method [Copyright &y& Elsevier]

Abstract: A quantitative method of capillary electrophoresis with sample stacking induced by moving reaction boundary (MRB) was developed for sensitive determination of oxymatrine (OMT) and matrine (MT) in rat plasma. The experimental conditions were optimized firstly. Below are the optimized experimental conditions: 20mM sodium formate solution (HCOONa, adjusted to pH 10.70 by ammonia) as sample solution, 3min 14mbar sample injection, 40mM formic buffer (HCOOH–HCOONa, pH 2.60) as stacking buffer, 7min 14mbar injection of stacking buffer, 100mM HCOOH–HCOONa (pH 4.80) as separation buffer, 73cm capillary (effective length 64cm), 21kV voltage, 210nm wavelength. Under the optimized conditions, higher than 60-fold sensitivity improvement of the stacking was simply achieved as compared with capillary zone electrophoresis, and the detectable limits obtained for OMT and MT were 0.26 and 0.19μgmL−1, respectively. Then, numerous demonstrations were carefully performed for the methodological validations of OMT and MT in rate plasma, including high specificity of method, good linearity (r =0.9993 for OMT, r =0.9991 for MT), fair wide linear concentration range (1.30–65.00μgmL−1 for OMT, 0.84–42.00μgmL−1 for MT), low limit of detection (1.03μgmL−1 for OMT, 0.38μgmL−1 for MT), less than 5% intra- and inter-day variance value, and higher than 96% recovery of OMT and MT in plasma. The developed method could be used for the trace analyses of OMT and MT in plasma and was finally used for the investigation on pharmacokinetic study of OMT in rat plasma. [Copyright &y& Elsevier]

Abstract: An on-line stacking method based on moving reaction boundary (MRB) was developed for the sensitive determination of barbital and phenobarbital in human urine via capillary electrophoresis (CE). The optimized conditions for the method are: 60mmolL−1 pH 11.0 Gly–NaOH as the background electrolyte, 10mmolL−1 pH 5.5 Gly–HCl as sample buffer, secobarbital as the internal standard (IS), 12.5kV, 1.4psi 10s sample injection, 75μm ID 60.2cm total length (50cm effective length) capillary and 214nm detect wavelength. Under the optimized conditions, the method can well stack and separate barbital and phenobarbital in urine samples and result in 20.5-fold and 22.6-fold improvement in concentration sensitivity for barbital and phenobarbital, respectively. Furthermore, the method holds: (1) good linear calibration functions for the two target compounds (correlation coefficients r >0.999), (2) low limits of detection (0.27μgmL−1 for barbital and 0.26μgmL−1 for phenobarbital), (3) low limits of quantification (0.92μgmL−1 for barbital and 0.87μgmL−1 for phenobarbital), (4) good precision (R.S.D. of intra-day and inter-day less than 5.38% for barbital and 1.67% for phenobarbital, respectively) and (5) high recoveries at three concentration levels (90.27–106.36% for barbital and 93.05–113.60% for phenobarbital in urine). The method is simple, sensitive and efficient, and can fit to the need of clinical and forensic toxicology. [Copyright &y& Elsevier]