Weak Cation Exchange Research Articles

Comprehensive Profiling of the Low Molecular Weight Proteins and Peptides in Weak Cation Exchange Beads Human Serum Retentate

Mass spectrometric profiling using ProteinChip and magnetic beads has rapidly grown over the past years, particularly to generate serum profiles for cancer diagnosis. The molecular weights of these distinguishing peaks are usually under 30 kDa. To identify those low molecular weight proteins and peptides is important for specific assays to be developed and increases biological insight. In this study, low molecular weight proteins and peptides from serum were purified by a combination of weak cation exchange magnetic beads and high performance liquid chromatography. The purified proteins and peptides were analyzed by 1D SDS PAGE, SELDI and LC-MS/MS. 246 proteins were identified from the HPLC fractions by LC-MS/MS. 95(38.62%) proteins were first identified in serum compare with Sys-BodyFluid database. 11(11/96) proteins were documented cancer associated proteins. We also observed about 109 proteins/peptides in SELDI mass spectrum, and 13 of the SELDI features were identified.

Proteomic analysis of urinary upper gastrointestinal cancer markers

We have investigated the use of human urine as a non-invasive medium to screen for molecular biomarkers of carcinomas of the upper gastrointestinal (uGI) tract using SELDI-TOF-MS. A total of 120 urine specimens from 60 control and 60 uGI cancer patients were analysed to establish a potential biomarker fingerprint for the weak cation exchanger CM10 chip surface, which was validated by blind testing using a further 59 samples from 33 control and 26 uGI cancer patients. Using Biomarker Pattern software, we established a model with a sensitivity of 98% and specificity of 95% for the learning sample set, and a sensitivity of 96% and specificity of 72% for the validation data set. Model variable importance included six peptides with m/z of 10,230, 10,436, 10,574, 10,311, 10,467, and 10,118 of which the 10, 230 molecular species was the main decider (sensitivity 86% and specificity 80%). Initial protein database searching identified 10,230 as S100-A6, 10,436 as S100-P, 10,467 as S100-A9, and 10,574 as S100-A12 of which S100-A6 and S100-A9 were confirmed by Western blotting. We have demonstrated that SELDI-TOF-MS as a screening tool is a rapid and valid methodology in the search for urinary cancer biomarkers, and is potentially useful in defining and consolidating biomarker patterns for uGI cancer screening.

Analysis of synovial fluid in knee joint of osteoarthritis:5 proteome patterns of joint inflammation based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The purpose of this study was to use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in osteoarthritis research. Our aim was to find differentially expressed disease-related and condition-specific peptide in synovial fluid in the knee joint of patients suffering from osteoarthritis (OA), and to develop and validate the peptide classification model for OA diagnosis. Based on the American College of Rheumatology criteria, 30 OA cases and ten healthy donors were enrolled and underwent analysis. Magnetic beads-based weak cation exchange chromatography (MB-WCX) was performed for sample processing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was conducted for peptide profile. ClinProt software 2.2 was used for data analysis and a genetic algorithm was created for class prediction. Two peptide peaks were found which may be characterised as the potential diagnostic markers for OA. Two other significantly different peptide peaks were found in OA patients at a medium stage compared to the early and late stages. A genetic algorithm (GA) was used to establish differential diagnosis models of OA. As a result, the algorithm models marked 100% of OA, and of 97.92% of medium-stage OA. This study demonstrated that use of proteomics methods to identify potential biomarkers of OA is possible, and the identified potential biomarkers may be potential markers for diagnosis and monitoring the progression of OA.

Determination of paraquat residue in plant-derived foodstuffs by high performance liquid chromatography-tandem mass spectrometry

A sensitive and selective method is presented for the determination of paraquat residue in fruits, vegetables, beans and grain by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Paraquat in samples was extracted with water and cleaned-up with a weak cation exchange (WCX) column to obtain an extract suitable for analysis using HPLC-MS/MS. The paraquat was separated by a CAPCELL PAK ST column (150 mm x 2.0 mm) and with acetonitrile-10 mmol/L ammonium acetate solution (adjusted to pH 4.0 by formic acid) as the mobile phase, and multiple reaction monitoring (MRM) was used with electrospray ionization in the positive ion mode. The calibration curve was linear between the peak area and the mass concentration of paraquat from 0.01 to 0.1 mg/L with the correlation coefficient of 0.993. Recoveries of paraquat spiked in samples at three levels ranged from 84.0% to 106.0% with the relative standard deviations of 7.8% - 18.8%. The limits of detection (LODs) of paraquat were 0.01 mg/kg in fruits and vegetables and 0.05 mg/kg in beans and grain. The LODs can meet the requirements of international maximum residue limit.

Preparation of poly(vinyltetrazole) chain-grafted poly(glycidymethacrylate-co-ethylenedimethacrylate) beads by surface-initiated atom transfer radical polymerization for the use in weak cation exchange and hydrophilic interaction chromatography

A novel stationary phase for weak cation exchange (WCX) and hydrophilic interaction chromatography (HILIC) was prepared with surface-initiated atom transfer radical polymerization (SI-ATRP). Vinyltetrazole was grafted onto the surface of the beads in water medium with the polyglycidylmethacrylate beads (PGMA/EDMA) previously modified with 2-bromoisobutryl bromide as the macromolecule initiators and CuCl as catalyst. The poly(vinyltetrazole)-grafted beads obtained with different atom transfer radical polymerization (ATRP) formulations were tried as chromatographic packings in ion-exchange chromatography. The results showed that the prepared columns could separate the tested proteins with high efficiency and high capacity, and the retention time of protein had a positive relationship with increasing the chain lengths of the grafted poly(vinyltetrazole) (PVT). The prepared column was also found to be able to separate nucleosides by hydrophilic interaction chromatographic mode.

The disposition of (R)-alpha-methylhistamine, a histamine H3-receptor agonist, in rats.

Using a modified HPLC method with a fluorescence spectrophotometer and a weak cation exchanger, it was possible to separate (R)-alpha-methylhistamine (alpha-methylhistamine) from histamine in plasma and various tissues. The assay was used to study the disposition and pharmacokinetic analysis of alpha-methylhistamine after a bolus intravenous administration to rats. After rapid intravenous administration (12.6 mg kg-1), the plasma concentration declined biexponentially with a half-life of 1.3 min in the elimination phase. The area under the plasma concentration-time curve and total body clearance were 130 micrograms min mL-1 and 97 mL min-1 kg-1, respectively. After administration, alpha-methylhistamine was immediately transferred to various tissues. The concentration was high in the kidney, lung, and liver (kidney > lung > liver), but low in the brain. The tissue-to-plasma concentration ratios in peripheral tissues were greater than 1, suggesting that the transfer of alpha-methylhistamine to peripheral tissues was due to a specialized transport mechanism or possibly to tissue binding. However, the finding that the tissue/plasma ratio in the brain was lower than unity suggests that the transport system in this tissue depends on a concentration gradient, and that alpha-methylhistamine crosses the blood-brain barrier in rats with difficulty.

STUDY ON THE RETENTION OF GLYCINEBETAINE ON A WEAK ACID CATION EXCHANGE RESIN

Weak acid cation (WAC) exchange resin in monovalent form is used in the recovery of glycinebetaine (GB) under alkaline conditions. The resin can also be used as a stationary phase at pH below the resin's pKa value, that is, in hydrogen form, when the retention behavior of GB is essentially different. In this study, the retention mechanism of GB was investigated on a WAC exchange resin in hydrogen, sodium, and intermediate ionic forms and with three other components: glycerol, choline chloride, and glycine. The experiments were carried out in a pilot scale column with acrylic acid-divinylbenzene gel type resin with aqueous eluent at neutral and acidic pH. GB was reported to retain notably longer on a hydrogen form WAC resin than on a corresponding sodium form exchange resin. Similar changes in retention time were seen for glycine, but not for glycerol or choline chloride. It was concluded that the retention mechanism of GB on the hydrogen form WAC exchange resin is related to hydrogen bonds between the resin and zwitterionic GB. The greater retention of GB on hydrogen form WAC resin can be utilized, for example, in the recovery process of GB from acidic solutions, such as vinasse.

Bioconcentration study of Xinjunan in zebrafish

An experiment was carried out to determine the acute toxicity and bioconcentration factor of Xinjunan in zebrafish under semi-static test method. The result of the 96-h LC(50) values (0.31 mg/L), at 95% confidence limit, revealed that Xinjunan was highly toxic. Bioconcentration factor after 8 days exposure, 451.0 and 273.2, respectively, at two concentrations, were at medium bioconcentration range. To determine Xinjunan residues in water and fish, a method was developed by using a liquid-liquid distribution and a cationic exchange solid-phase extraction method to extract and clean up Xinjunan in fish, and then using a weak cationic exchange column with gradient elution and second-order mass spectrometry with selected reaction monitoring mode detection. This method found a good linear relationship (r > 0.99), the lowest limit of quantification with a signal-to-noise ratio of 10:1 was 0.02 [Formula: see text]g/L in water and 5 [Formula: see text]g/kg in fish, the recovery ranged from 97% to 109% for water and fish at different levels with a coefficient of variation less than 5%. The accuracy, precision, and lowest limit of detection of the method used for residue analysis of Xinjunan in water and fish can meet environmental exposure monitoring requirements. The results of the acute toxicity and bioconcentration provide a basis for environmental risk analysis of Xinjunan.

Preparation of High-capacity, Monodisperse Polymeric Weak Cation Exchange Packings Using Surface-initiated Atom Transfer Radical Polymerization and Its Chromatographic Properties

A novel stationary phase for weak cation exchange (WCX) chromatography was prepared by "grafting from" strategy. Surface initiated atom transfer radical polymerization (ATRP) of acrylic acid (AA) was conducted in toluene medium, starting from the macromolecule initiators of poly(4-vinylbenzyl chloride-co-divinylbenzene) (PCMS/DVB) beads. The amounts of poly(acrylic acid) grafted chains with different ATRP formulations were calculated based on the elemental analyses. The poly(acrylic acid) grafted beads obtained with different ATRP formulations were tried as chromatographic packings in the separation of proteins by ion-exchange chromatography. The effect of the poly(acrylic acid) grafted chain lengths on PCMS/DVB beads for the separation of proteins was investigated in details. Simultaneously, characterization of the column was investigated as ion chromatographic stationary phase for the separation of inorganic cations. The results show that poly(acrylic acid) grafted columns had excellent performance for separation of proteins and inorganic cations. The highest of the dynamic capacity of the column was 35.55 mg/mL. The columns were provided with high column efficiency.

Comparison of protein profiles between acetonitrile- and non-acetonitrile-treated sera from patients with nasopharyngeal carcinomas

Serum proteins may be abnormally increased or decreased during the occurrence and development of nasopharyngeal carcinoma (NPC). However, currently there are no simple or effective methods to collect and differentiate these abnormally secreted proteins from abundant serum proteins. In this study, acetonitrile was used to remove the majority of high-abundance proteins from serum samples obtained from patients with NPC. The samples were subjected to surface-enhanced laser desorption/ionization time-of-flight mass spectrometry with a CM10 (weak cation exchange) ProteinChip, and the resulting protein profiles were compared with those of non-acetonitrile-treated serum samples. The results showed that the protein profiles differed between the acetonitrile- and non-acetonitrile-treated sera from patients with NPC. A large proportion of the non-acetonitrile-treated NPC serum protein peaks were <6000 kDa, while the detection rate of protein peaks >6000 kDa was relatively higher in the acetonitrile-treated NPC sera, accounting for more than half of all protein peaks (26.2+37.5%). Few differentially upregulated proteins were lost, and the peak value density increased after acetonitrile treatment. In conclusion, acetonitrile treatment of serum samples is effective in removing high-abundance macromolecular proteins. Therefore, acetonitrile treatment can be applied for the investigation of serum proteomics and may aid in the identification of differentially expressed proteins.

Application of protein expression profiling to screen chemicals for androgenic activity

Protein expression changes can be used for detection of biomarkers that can be applied diagnostically to screen chemicals for endocrine modifying activity. In this study, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) coupled with a short term fish assay was used to investigate changes in plasma protein expression as a means to screen chemicals for androgenic activity. Adult gravid female sheepshead minnows ( Cyprinodon variegatus) were placed into separate aquaria for seawater control, ethanol solvent control, and the following androgen agonist treatments at 5.0 μg/L: dihydrotestosterone (DHT), methyldihydrotestosterone (MDHT), testosterone (T), methyltestosterone (MT) and trenbolone (TB). Treatments of 0.6 μg/L endosulfan and 40 μg/L chlorpyrifos (CP) served as non-androgenic negative stressor controls. Test concentrations were maintained using an intermittent flow-through dosing apparatus supplying exposure water at 20 L/h. Fish were sampled at 7 days, the plasma diluted, processed on weak cation exchange CM10 ProteinChip arrays and analyzed. Spectral processing resulted in 249 individual m/ z peak clusters for the androgen exposed fish. Partial least squares-discriminant analysis was used to develop an androgen-responsive model using sample spectra from exposures with DHT and unexposed solvent control fish as the training set. The androgen classification model performed with ≥79% specificity (% true negative) and ≥70% sensitivity (% true positive) for non-aromatizable androgens. The aromatizable androgens T and MT were classified as androgenic with specificities of 42 and 79%, respectively. The reduction in sensitivity observed with T is thought to be caused by its metabolic conversion to an estrogen by aromatase. The results of these studies show diagnostic plasma protein expression models can correctly classify chemicals by their androgenic activity using a combination of high throughput mass spectrometry and multivariate approaches.

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Combined with Magnetic Beads for Detecting Serum Protein Biomarkers in Parkinson’s Disease

Background: Biomarkers for neurodegenerative diseases are essential to facilitate disease diagnosis. Application of proteomics has greatly hastened the search for novel biomarkers. In this study, new potential biomarkers were discovered, and a diagnostic pattern was established for idiopathic Parkinson’s disease (PD) by using proteomic technology. Methods: Serum proteins from PD patients and controls were captured by magnetic bead-based weak cation exchange. The molecular weight of the proteins in bead-binding fraction was detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Biomarker Wizard 3.1 and Biomarker Patterns Software were used for data analysis and constructing a model of biomarkers. A blinded testing set was used to validate the model. Results: A total of 17 discriminating m/z peaks related to PD were identified. The model based on the 5 biomarkers generated an excellent separation between PD and healthy controls with 98.36% for the sensitivity and 83.05% for the specificity. Blind test data demonstrated the model could recognize patients with PD with a sensitivity of 85.0% and a specificity of 70.0%. Conclusions: The preliminary data suggested a potential application of MALDI-TOF-MS combined with magnetic beads. The model comprising 5 promising biomarkers can differentiate individuals with PD and the healthy subjects precisely.

Tracing the transition of methicillin resistance in sub-populations of Staphylococcus aureus, using SELDI-TOF Mass Spectrometry and Artificial Neural Network Analysis

Strains ( n = 99) of Staphylococcus aureus isolated from a large number of clinical sources and tested for methicillin sensitivity were analysed by MALDI-TOF-MS using the Weak Cation Exchange (CM10) ProteinChip Array (designated SELDI-TOF-MS). The profile data generated was analysed using Artificial Neural Network (ANN) Analysis modelling techniques. Seven key ions identified by the ANNs that were predictive of MRSA and MSSA were validated by incorporation into a model. This model exhibited an area under the ROC curve value of 0.9147 indicating the potential application of this approach for rapidly characterising MRSA and MSSA isolates. Nearly all strains ( n = 97) were correctly assigned to the correct group, with only two aberrant MSSA strains being misclassified. However, approximately 21% of the strains appeared to be in a process of transition as resistance to methicillin was being acquired.

Application of serum proteomic patterns for the detection of esophageal carcinoma in Kazakh in Xinjiang

To detect the serum protein profiles of Kazakh's esophageal carcinoma (EC) patients in Xinjiang by SELDI-TOF-MS (surface enhanced laser desorption & ionization time-of-flight mass spectrometry) and build up a diagnostic model of Kazakh's EC in Xinjiang. The serum samples from 41 Kazakh's EC patients and 20 Kazakh's healthy controls were collected and analyzed on weak cation exchange and hydrophobic surface protein chip by SELDI-TOF-MS technology. The differentially expressed markers of esophageal carcinoma were detected. The values of M/Zs were significantly different between Kazakh's EC patients and controls (P < 0.05). Among these, 6 proteins peaks were up-regulated (5495.2265, 15 964.6951, 16 152.0872, 4488.4818, 8164.7652, 4979.4223) and 4 down-regulated (6900.3285, 13 790.9241, 8790.8130, 8714.7915) in the Kazakh's EC group. According to cross validation, the model of Kazakh's EC made up of 7 proteins (M/Z 6900.3285, 13 790.9241, 8790.8130, 15 964.6951, 16 152.0872, 4488.4818, 4979.4223) was established. The sensitivity and specificity of this model were 100% (41/41) and 100% (20/20) respectively. The model with 7 proteins markers has a higher sensitivity and specificity for Kazakh's EC patients in Xinjiang. It may provide a new serum diagnostic tool for Kazakh's EC patients in Xinjiang.

Identify Biomarkers of Neuropsychiatric Systemic Lupus Erythematosus by Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Combined with Weak Cation Magnetic Beads

To identify proteomic biomarkers in cerebrospinal fluid (CSF) and develop a diagnostic proteomic model for neuropsychiatric systemic lupus erythematosus (NPSLE). CSF proteomic spectra were generated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) combined with weak cation exchange (WCX) magnetic beads. The spectra were taken from 27 patients with NPSLE before and after treatment, and 27 controls including 17 patients with scoliosis and 10 patients with SLE but without neuropsychiatric manifestation. Discriminating peaks were processed by Biomarker Patterns Software to build a decision tree model for NPSLE classification. In addition, CSF samples of 12 patients with NPSLE, 12 patients with lumbar disc herniation, and 9 patients with other neurological conditions were used as a blind test group to verify the accuracy of the model. Twelve discriminating mass-to-charge (m/z) peaks were identified between NPSLE and controls: m/z peaks 7740, 11962, 8065, 7661, 6637, 5978, 11384, 11744, 8595, 10848, 7170, and 5806. The diagnostic decision tree model, built with a panel of m/z peaks 8595, 7170, 7661, 7740, and 5806, recognized NPSLE with both sensitivity and specificity of 92.6%, based on training group samples, and sensitivity and specificity of 91.7% and 85.7%, respectively, based on the blind test group. In addition, the root node m/z peak 8595 protein, which was downregulated in the CSF of patients with NPSLE after treatment, was identified and confirmed as ubiquitin by immunoprecipitation and ELISA. Potential CSF biomarkers for NPSLE are identified by MALDI-TOF-MS combined with WCX magnetic beads. The novel diagnostic proteomic model with m/z peaks 8595, 7170, 7661, 7740, and 5806 is highly sensitive and relatively specific for NPSLE diagnosis. The level of ubiquitin in CSF is a promising biomarker for active NPSLE.

MALDI-TOF MS Combined With Magnetic Beads for Detecting Serum Protein Biomarkers and Establishment of Boosting Decision Tree Model for Diagnosis of Colorectal Cancer

The aim of present study is to study the serum protein fingerprint of patients with colorectal cancer (CRC) and to screen protein molecules that are closely related to colorectal cancer during the onset and progression of the disease with Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Serum samples from 144 patients with CRC and 120 healthy volunteers were adopted in present study. Weak cation exchange (WCX) magnetic beads and PBSII-C protein chips reader (Ciphergen Biosystems Ins.) were used. The protein fingerprint expression of all the Serum samples and the resulted profiles between cancer and normal groups were analyzed with Biomarker Wizard system. Several proteomic peaks were detected and four potential biomarkers with different expression profiles were identified with their relative molecular weights of 2870.7 Da, 3084 Da, 9180.5 Da, and 13748.8 Da, respectively. Among the four proteins, two proteins with m/z 2870.7 and 3084 were down-regulated, and the other two with m/z 9180.5 and 13748.8 were up-regulated in serum samples from CRC patients. The present diagnostic model could distinguish CRC from healthy controls with the sensitivity of 92.85% and the specificity of 91.25%. Blind test data indicated a sensitivity of 86.95% and a specificity of 85%. The result suggested that MALDI technology could be used to screen critical proteins with differential expression in the serum of CRC patients. These differentially regulated proteins were considered as potential biomarkers for the patients with CRC in the serum and of the potential value for further investigation.

Proteomic technologies for identification of serum biomarkers of potential autoimmune demyelinating polyneuropathies

Time-of-flight MALDI mass spectrometry (MALDI-TOF-MS) profiling of blood serum of patients with Guillain-Barré syndrome (GBS, 36 samples), chronic inflammatory demyelinating polyneuropathy (CIDP, 24 samples) and practically healthy donors (HD) (35 samples) was carried out in order to identify potential biomarkers of autoimmune demyelinating polyneuropathies (ADP). To simplify the peptide-protein mixture of serum prior to MALDI-TOF-MS analysis samples were pre-fractionated on magnetic microparticles with a weak cation-exchange (MB-WCX) surface. Comparative analysis of mass spectrometric data using the classification algorithms (genetic and neural network-controlled) revealed a characteristic set of peaks, agreed change area with a high specificity and sensitivity of the differentiated mass spectrometry profiles of the blood serum of patients with DPNP and healthy donors (for GBS values of these characteristics reached 100 and 100, and for CIDP 94.1 and 100% respectively). Comparative analysis of mass spectrometric profiles of serum samples obtained from patients with GBS and CIDP, allowed to build a classification model to differentiate these diseases from each other, with a specificity of 88.9 and a sensitivity of 80%.

Analysis of the urinary peptidome associated withHelicobacter pyloriinfection

To investigate the relationship between urinary peptide changes and Helicobacter pylori (H. pylori) infection using urinary peptidome profiling. The study was performed in volunteers (n=137) who gave informed consent. Urinary peptides were enriched by magnetic beads based weak cation exchange chromatography and spectrums acquired by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). ClinProTools bioinformatics software was used for statistical analysis and the recognition of peptide patterns. The marker peptides were identified by LTQ Obitrap XL tandem MS. Approximately 50 proteins or peptides which loaded onto the magnetic beads were detected by MALDI-TOF MS. By optimizing the parameters of the model, the Genetic Algorithm model had good recognition capability (97%) and positive predictive value (94%). Based on the model, 2 markers with molecular masses of 6788 and 1912 Da were found that differentiated between H. pylori positive and negative volunteers. The m/z 1912 sequence was parsed as SKQFTSSTSYNRGDSTF. The peptide was identified as isoform 1 of the fibrinogen α chain precursor, whose concentration in urine was markedly higher in H. pylori infected volunteers than in H. pylori non-infected ones. The appearance of urinary fibrinogen degradation products is caused by an active H. pylori-induced process.

Rapid quantification of tilidine, nortilidine, and bisnortilidine in urine by automated online SPE-LC-MS/MS

The opioid tilidine is a prodrug which is hepatically metabolized to active nortilidine and bisnortilidine. Due to the increasing abuse of tilidine by drug users and the lack of a specific immunoassay, we developed an analytical method for the quantification of tilidine, nortilidine, and bisnortilidine in urine suitable for screening. In a following step, this method was used to establish data on excretion kinetics of the substances in order to evaluate the time window of detection after a single oral dose of tilidine/naloxone and also was applied to authentic urine samples from correctional facilities. Urine samples were mixed with internal standard solution and extracted on a weak cation exchanger at pH 6 using a Symbiosis Pico system. The chromatographic separation was achieved within a 3.5-min run time on a Phenylhexyl column (50 × 2.0 mm, 5 μm) via gradient elution (methanol and 0.2% formic acid) at a flow rate of 0.50 mL/min. The ESI-MS/MS was performed on a QTrap 3,200 in positive multiple reaction monitoring mode using two mass transitions per analyte. Validating the method resulted in a lower limit of quantification of 1.0 μg/L followed by a linear calibration range to 100 μg/L for each analyte (r(2) > 0.99). The analytical method allowed the detection of a single dose of a commercially available tilidine solution up to 7 days after administration. Using this highly sensitive method, 55 of 3,665 urine samples were tested positive.

At-line coupling of magnetic-nanoparticle-based extraction with gel isoelectric focusing for protein analysis

Sample preparation is a crucial step for protein analysis. Functionalized magnetic nanoparticle (MNP)-based extraction has been developed to be a useful sample preparation technique for proteomic analysis. In this paper, we present a strategy for at-line coupling of MNP-based extraction (MNE) with gel isoelectric focusing (IEF). The key to the at-line combination is to use an anolyte or a catholyte as the desorbing agent. Thus, functionalized MNPs can be facilely at-line coupled with gel IEF, provided that the extraction/desorption process is pH-controlled. MNPs extracted with target proteins are added to the sample well, which can function as a natural adapter. Once a focusing electric field has been applied across the gel, proton ions migrating from the anolyte or hydroxide ions migrating from the catholyte can act as a desorbing agent, releasing the proteins from the MNE probes. The released proteins are consequently focused into distinct bands where the local pH equals their pI values. The at-line combination was well demonstrated with three types of functionalized nanoparticles: (1) phenylboronic acid functionalized MNPs for extracting glycoproteins through boronate affinity; (2) carboxyl-functionalized MNPs for extracting positively charged proteins through a weak cation exchange mechanism; and (3) amino-functionalized MNPs for extracting negatively charged proteins through a weak anion exchange mechanism. The at-line combination exhibited several significant advantages, including selectivity, sensitivity, and speed.