Enrichment Column Research Articles

Nutrient status, plant availability and seasonal forcing mediate fish herbivory in temperate seagrass beds

Among mechanisms for herbivore choice, palatability and abundance of food resources appear to have a central role in foraging ecology. Hence, nutrient supply due to eutrophication may cause profound alterations to both nutritional and quantitative aspects of primary producers, leading to changes in herbivory pressure and enhancing overall functional changes. We present an experi- ment of water column enrichment in an oligotrophic seagrass bed of Posidonia oceanica (northwest- ern Mediterranean Sea). In this study we measured monthly seasonal changes in fish herbivore behavior following alterations in qualitative (nutrient content, species composition) and quantitative (biomass availability) features of primary producers (seagrass, epiphytes and macroalgae) as a result of nutrient fertilization. Nutrient addition induced changes in both seagrass (enhanced plant N con- tent and leaf growth) and epiphytes (enhanced N content, biomass load and altered species compo- sition) throughout late spring and summer. This nutritional alteration of food organoleptic properties concurred within the seasonal peak of herbivory by the fish Sarpa salpa and caused a disproportion- ate herbivore pressure on enriched plots, until shoots were reduced to around 50% of their original leaf area. When plant biomass on fertilized plots was substantially reduced, herbivores fed mostly on control plots until the end of the summer period. During the rest of the year (autumn and winter), moderate nutrient additions could not disrupt the seasonal inertia of the ecosystem. Hence, our results show evidence that the foraging behavior of herbivorous fish is the result of chemical and visual sensing of the environment. Seasonal forcing, however, tightly controls ecosystem processes and the biological cycles of species, adding a further step to functional alterations in temperate sea- grass meadows subjected to eutrophication.

Micro near infrared spectroscopy (MicroNIRS) based on on-line enrichment: Determination of trace copper in water using glycidyl methacrylate-based monolithic material

A micro near infrared spectroscopy (MicroNIRS) technique based on monolithic column enrichment has been developed to detect low concentration metallic ions in water. Glycidyl methacrylate–ethylene dimethacrylate–iminodiacetic acid (GMA–EDMA–IDA) monolithic material was synthesized to construct an enrichment device, which can enrich metallic ions more efficiently than chelating resin materials such as D401. Cu 2+ solutions passed through the enrichment device and concentrated on the surface of the material, and then diffuse reflection NIR (DRNIR) spectra were measured by touching the NIR probe on the surface. The spectra were pretreated with multiplicative scatter correction (MSC) and the MSC-pretreated spectra showed high linear correlation to the Cu 2+ concentrations in spectral regions of 900–1350 nm, 1420–1480 nm and 1970–2050 nm for all concentration ranges. PLS models built with leave-one-out cross-validation also yielded good features. The mean relative error (MRE) of prediction was 10.0% in the 0.0001–0.0010 μg mL −1 range. The concentration has low to ppb-level, but the MRE was not more than 10%, which was acceptable for trace analysis. The results revealed that the MicroNIRS technique enhanced the sensitivity of NIRS dramatically and widened the NIRS technique to micro and trace analysis field.

Improvement in deuterium recovery from water–isotopes mixture by thermal diffusion in the concentric-tube device of branch columns

The remixing effect due to the forced convection-currents in a continuous-flow concentric-tube thermal-diffusion column for enrichment of deuterium from water–isotopes mixture can be reduced by employing the device of branch columns, instead of the device of single column, with the same total column length. The improvement in deuterium recovery increases when the flow rate or the total column length increases. It is found that the improvement can reach about 50% for the numerical example given.

Characterization and evaluation of two-dimensional microfluidic chip-HPLC coupled to tandem mass spectrometry for quantitative analysis of 7-aminoflunitrazepam in human urine

Microfluidic chip-based high-performance-liquid-chromatography coupled to mass spectrometry (chip-HPLC-MS) has been widely used in proteomic research due to its enhanced sensitivity. We employed a chip-HPLC-MS system for determining small molecules such as drug metabolites in biological fluids. This chip-HPLC-MS system integrates a microfluidic switch, a 2-dimensional column design including an enrichment column (160 nL) for sample pre-concentration and an analytical column for chromatographic separation, as well as a nanospray emitter on a single polyimide chip. In this study, a relatively large sample volume (500 nL) was injected into the enrichment column for pre-concentration and an additional 4 μL of the initial mobile phase was applied to remove un-retained components from the sample matrix prior to chromatographic separation. The 2-dimensional column design provides the advantages of online sample concentration and reducing matrix influence on MS detection. 7-Aminoflunitrazepam (7-aminoFM2), a major metabolite of flunitrazepam (FM2), was determined in urine samples using the integrated chip-HPLC-MS system. The linear range was 0.1-10 ng mL(-1) and the method detection limit (signal-to-noise ratio of 3) was 0.05 ng mL(-1) for 7-aminoFM2. After consecutive liquid-liquid extraction (LLE) and solid-phase extraction (SPE), the chip-HPLC-MS exhibited high correlation between 7-aminoFM2 spiked Milli-Q water and 7-aminoFM2 spiked urine samples. This system also showed good precision (n = 5) and recovery for spiked urine samples at the levels of 0.1, 1.0, and 10 ng mL(-1). Intra-day and inter-day precision were 2.0-7.1% and 4.3-6.0%, respectively. Clinical urine samples were also analyzed by this chip-HPLC-MS system and acceptable relative differences (-1.3 to -13.0%) compared with the results using a GC-MC method were determined. Due to its high sensitivity and ease of operation, the chip-HPLC-MS system can be utilized for the determination of small molecules such as drug metabolites and neurotransmitters in biological fluids for clinical diagnosis.

Immunomagnetic nanoparticle-based sandwich chemiluminescence-ELISA for the enrichment and quantification of E. coli

Rapid methods for the quantification of Escherichia coli are required for the monitoring of faecal contamination in water to secure public health. The immunomagnetic separation (IMS) offers rapid enrichment and purification of bacteria in complex matrices and is compatible with immunoassays. By means of this technique, non-target cells and matrix components which might interfere with subsequent analytical methods are removed. We present the synthesis of magnetic nanoparticles (MNPs) and covalent coupling to antibodies against the enterobacterial common antigen (ECA) for use with IMS. Quantification was carried out with a chemiluminescence-based sandwich enzyme-linked immunosorbent assay (ELISA). Our anti-ECA-MNPs allow for a group-specific enrichment of bacterial cells, which can be combined with a species-specific analytical method. The particles were used along with commercially available magnetic columns for the selective enrichment of E. coli from 10-mL water samples. The volumetric enrichment factor was 9. For enriched samples, the limit of detection was reduced from 5.0 × 106 cells·mL-1 to 2.6 × 105 cells·mL-1. Using 200 µL anti-ECA-MNPs, we determined a recovery of 97 ± 6% for a sample containing 106 cells·mL-1 and 89 ± 2% for a sample containing 107 cells·mL-1. The overall time for cell enrichment and detection was 3 h 45 min.

The optimum number of columns for enrichment of heavy water by thermal diffusion in the Frazier scheme at fixed sum of column heights

The effect of the number of columns on the enrichment of heavy water from water isotope mixture by thermal diffusion in the Frazier scheme at fixed sum of column heights has been investigated. The equations for predicting the optimum number of columns and the corresponding maximum separation have been derived. Considerable enrichment in separation is obtainable if the number of columns, as well as the column height, in a Frazier scheme is properly assigned for a certain flow-rate operation.

Development and validation of a sensitive assay for the quantification of imatinib using LC/LC‐MS/MS in human whole blood and cell culture

We developed and validated a semi-automated LC/LC-MS/MS assay for the quantification of imatinib in human whole blood and leukemia cells. After protein precipitation, samples were injected into the HPLC system and trapped onto the enrichment column (flow 5 mL/min); extracts were back-flushed onto the analytical column. Ion transitions [M + H](+) of imatinib (m/z = 494.3 --> 394.3) and its internal standard trazodone (372.5 --> 176.3) were monitored. The range of reliable response was 0.03-75 ng/mL. The inter-day precisions were: 8.4% (0.03 ng/mL), 7.2% (0.1 ng/mL), 6.5% (1 ng/mL), 8.2% (10 ng/mL) and 4.3% (75 ng/mL) with no interference from ion suppression. Autosampler stability was 24 hs and samples were stable over three freeze-thaw cycles. This semi-automated method is simple with only one manual step, uses a commercially available internal standard, and has proven to be robust in larger studies.

Selective and Sensitive Determination of Mercury(II) Ions in River Water by an Automatic HPLC System Combined with On-Line Column Enrichment as 2-Mercaptopyrimidine Chelate

A selective and sensitive determination method for mercury (Hg) ions in river water by reversed phase high performance liquid chromatography (HPLC) has been developed. Two mL of water sample containing Hg(II) ions was put into a 4 mL sample tube. To the sample tube, 500 µL of 2 mol/L (M) acetate buffer solution (pH 4.5), and 50 µL of 0.01 M 2-mercaptopyrimidine (MP) were added to form a neutral Hg chelate. The Hg chelate subsequently became preconcentrated in a reversed phase mini column. By switching the valve, the Hg chelate was separated and detected at 255 nm. These processes occur automatically, except for the addition of water samples, buffer, and MP solutions. The correlation coefficients of the calibration curves obtained with 2 mL of mercury standard were more than 0.999 over the range of 20 ng/mL (ppb) to 10 µg/mL (ppm). The detection limit of Hg ions in 2 mL solution was 1.6 ppb, which corresponded to 3 times the standard deviation (N = 10) of the blank peak area. Effects of foreign ions on the determination of 0.2 ppm Hg were investigated with 57 metal ions. Almost none of the ions interfered except for Au(III), Pd(II), Ag(I), and Co(II) ions. The recoveries with spiked river water samples for 5, 0.5, and 0.05 Hg ion (N = 7) were 99.7 ± 0.4%, 100.3 ± 0.3%, and 100.7 ± 0.7%, respectively.

Experimental manipulation of sediment organic content and water column aeration reduces Zostera marina (eelgrass) growth and survival

The effect of synergy between sediment organic enrichment and lack of night oxygen renewal in the water column on growth and survival of Zostera marina, and how it is reflected in the sulfur parameters in the plants ( δ 34 S, TS and S 0) was studied experimentally. An experiment consisting of Z. marina mesocosms with different levels of organic enrichment and water column aeration was established, and the effects on sediment conditions, sulfide invasion and growth and survival of Z. marina were examined over a 4 week period. Shoots growing in Ambient Organic matter–sediments showed signs of sulfide invasion, as TS increased in all plant compartments and δ 34 S of the plant tissues decreased during the experiment, but neither growth rate nor survival were significantly affected. The lack of night oxygen renewal had no evident effects in non-enriched sediments as porewater sulfide concentrations, AVS- and CRS-pools were not different from the corresponding 24 h aeration treatment. Plant growth rate and survival were neither different from the corresponding 24 h aeration treatment. On the contrary, shoots growing on High Organic matter-sediments suffered a massive sulfide invasion and it was directly correlated to the observed decrease in growth rates. Even though the lack of night oxygen renewal had no evident effects on sediment variables there were, however, strong indications that the different aeration levels affected plant performance, suggesting a lower sulfide oxidation capacity and confirming that low water column oxygen concentrations reduces the defense capacity of the shoots against sulfide invasion. Although δ 34 S, TS and S 0 concentrations together provided a powerful set of indicators to detect the invasion of sulfide in Z. marina shoots, this study enlightens the need for a deeper investigation of sulfide intrusion in seagrasses and the relation between plant sulfur parameters and sediment conditions.

Profile of native N‐linked glycan structures from human serum using high performance liquid chromatography on a microfluidic chip and time‐of‐flight mass spectrometry

Protein glycosylation involves the addition of monosaccharides in a stepwise process requiring no glycan template. Therefore, identifying the numerous glycoforms, including isomers, can help elucidate the biological function(s) of particular glycans. A method to assess the diversity of the N-linked oligosaccharides released from human serum without derivatization has been developed using on-line nanoLC and high resolution TOF MS. The N-linked oligosaccharides were analyzed with MALDI FT-ICR MS and microchip LC MS (HPLC-Chip/TOF MS). Two microfluidic chips were employed, the glycan chip (40 nL enrichment column, 43 x 0.075 mm(2) i.d. analytical column) and the high capacity chip (160 nL enrichment column, 140 x 0.075 mm(2) i.d. analytical column), both with graphitized carbon as the stationary phase. Both chips offered good sensitivity and reproducibility in separating a heterogeneous mixture of neutral and anionic oligosaccharides between injections. Increasing the length and volume of the enrichment and the analytical columns improved resolution of the peaks. Complex type N-linked oligosaccharides were the most abundant oligosaccharides in human serum accounting for approximately 96% of the total glycans identified, while hybrid and high mannose type oligosaccharides comprise the remaining approximately 4%.

Enrichment of protein–RNA crosslinks from crude UV‐irradiated mixtures for MS analysis by on‐line chromatography using titanium dioxide columns

UV crosslinking is an appropriate method to identify proteins that directly contact nucleic acid, e.g., RNA. In combination with modern mass spectrometric (MS) analysis such an approach provides the opportunity to reveal not only the nature of the crosslinked proteins but also to identify the actual crosslinking sites between the protein and the nucleic acid. However, the relatively low yield in UV-induced crosslinking makes it difficult to identify in particular those species by MS that represent peptide-nucleic acid conjugates, as the great excess of noncrosslinked material interferes with their detection in MS. Here, we present an automated enrichment strategy of crosslinked peptide-RNA oligonucleotides derived from crude mixtures of UV-irradiated ribonucleoprotein (RNP) particles that uses TiO(2) columns integrated within a two-dimensional (2D) nanoliquid chromatography (LC) system. The setup combines two C18 precolumns, a TiO(2) enrichment column and a nanoanalytical column. It allows the removal of the noncrosslinked RNA and protein moiety and the specific enrichment of crosslinked peptide-RNA conjugates so that UV-irradiated and subsequently completely hydrolyzed RNP complexes can directly be loaded and analyzed by MS. In this feasibility study, we demonstrate the specific enrichment of peptide-RNA oligonucleotides derived from UV-irradiated native spliceosomal U1 snRNPs and spliceosomal [15.5K-61K-U4atac snRNA] complex reconstituted in vitro.

A comparison of apple cultivars regarding ethylene production and physico-chemical changes during cold storage

Measurements of titratable acidity, soluble solids, firmness, ethylene production and weight loss were made for five apple cultivars held in cold storage for 100 days. Carbosieve G in the traps of the enrichment column, which has only a moderate affinity for light hydrocarbons, was found to meet the requirements for the optimal thermal desorption of ethylene (130°C for 2 minutes) from the enrichment column to the analytical column. ANOVA showed significant differences in all these five parameters between the five cultivars Golden Delicious Reinders, Resista, Topaz, Meteor and Rubinstep, and also in the course of storage. In all cases, the changes in titratable acids measured during storage were especially significant, but the observed changes in sugar levels, as measured by refractometry, were too variable to be useful in this context. High rates of ethylene production impacted probably only indirectly on the loss of firmness and the other parameters which were measured. Discriminant analysis of the measurements of firmness, ethylene production and titratable acid provided the best means of differentiating the cultivars, although Golden Delicious Reinders and Resista still could not be completely separated. Other parameters (soluble solids and loss in weight) did not contribute to the discriminant resolution.

Deoxynivalenol: Rationale for development and application of a urinary biomarker

Mycotoxins are common dietary contaminants in most regions of the world. The frequency of exposure to the various families of mycotoxins is often dependent on geographic location, national wealth and related agricultural and regulatory infrastructure, combined with diversity of diet and degree of food sufficiency. Deoxynivalenol (DON) is a Fusarium mycotoxin that frequently contaminates wheat, corn and barley in temperate regions. A number of acute poisoning incidences have been linked to DON-contaminated foods and chronic exposure to lower levels of DON has been predicted in many regions. DON is a potent animal toxin and exposure in humans may cause gastroenteritis, growth faltering and immune toxicity. An ability to conduct accurate exposure assessment at the individual level is required to fully understand the potential health consequences for humans. To date, such exposure biomarkers have been lacking for many important mycotoxins, including DON. To better assess exposure to DON at the individual level, we have developed a robust urinary assay, incorporating immunoaffinity column (IAC) enrichment and LC–MS detection. Further refinement of this urinary assay, by inclusion of 13C-DON as an internal standard, was then undertaken and tested within the UK. DON was frequently observed in urine and was significantly associated with cereal intake. A dietary intervention study demonstrated that avoiding wheat in the diet markedly reduced urinary levels of DON. This biomarker requires further validation but our initial data suggest it may provide a useful tool in epidemiological investigations of the potential health consequences of this common environmental toxin.

Hyper-phosphorylation of α-enolase in hypertrophied left ventricle of spontaneously hypertensive rat

Cardiac hypertrophy is one of the main target organ damages of essential hypertension and predicts a poor prognosis of the disease. The molecules involved in this event, especially their posttranslational modifications, remain largely unknown to date. With a combination of phosphoprotein column enrichment and two-dimensional gel electrophoresis separation, here we compared the profiling of enriched phosphoproteins from the left ventricle (LV) of spontaneously hypertensive rats (SHR) to that of age-matched Wistar Kyoto rats. As a result, 19 differential proteins were found in the hypertrophied LV of SHR. Among them, we focused on a glycolysis enzyme α-enolase, of which the hyper-phosphorylation was shown in the hypertrophied LV but not in non-hypertrophied atrium and right ventricle of SHR. Furthermore, the α-enolase hyper-phosphorylation was accompanied by decreased enzymatic activity. The further investigation based on these results would provide new clues to understand the pathological process of cardiac hypertrophy in SHR.

Nutrient effects on intertidal community: from bacteria to invertebrates

This study investigated the impacts of nutrient enrichment on intertidal biofilm micro- bial communities and on larval metamorphosis of invertebrates. We modulated the nutrient levels in the water column during biofilm development and examined the resulting biofilms for bacterial and diatom community composition using 2 DNA fingerprinting techniques and microscopic examina- tion, respectively. Results of both terminal-restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) analyses indicated that the nutrient enrichment treatment altered the biofilm bacterial communities relative to the control. Biofilms that had different microbial compositions were tested for their effects on larval metamorphosis of the polychaete Hydroides elegans, the barnacle Balanus amphitrite, and the slipper limpet Crepidula onyx. We showed that nutrient enrichment in the water column effectively changed the biofilm composition in ways that increased its inductiveness for larval metamorphosis of C. onyx and B. amphitrite, but not of H. elegans. This study highlights the apparent importance of nutrient enrichment in influencing the intertidal biofilm composition directly and the invertebrates indirectly.

Improving the resolution of neuropeptides in rat brain with on‐line HILIC‐RP compared to on‐line SCX‐RP

Our two already established on-line 2-D LC systems, a strong cation exchange-RP chromatography (SCX-RP) system and a hydrophilic interaction LC (HILIC)-RP 2-D LC system, were compared to explore which system is best suited for our further studies of differences in cerebral neuropeptide expression as a function of hypoxia-caused stress. The same mass spectrometer and database search parameters were applied in both systems. In total, 19 first dimension fractions were collected with the novel on-line HILIC-RP system, including a Hypercarb SPE column that was applied to trap the compounds not retained on a Kromasil C18 enrichment column. In contrast, six fractions were collected in the SCX-RP method, due to practical limitations of this traditional on-line 2-D LC system. With the on-line HILIC-RP system three times more peaks were detected. It was observed that most of the compounds eluted in the first two fractions in the SCX-RP method, while in the 2-D HILIC-RP method there seemed to be no correlation between peaks detected and fraction number. Thus, from this systematic study it seems that on-line HILIC-RP chromatography is the method of choice for comparative peptidomics of cerebral neuropeptides in future studies.

Sensitive determination of a glyoxal–DNA adduct biomarker candidate by column switching capillary liquid chromatography electrospray ionization mass spectrometry

A method based on column switching packed capillary liquid chromatography electrospray mass spectrometry has been developed for the determination of the adduct glyoxal-deoxyguanosine, a biomarker candidate for the assessment of glyoxal exposure, in DNA hydrolysate solutions. Microgram amounts of DNA were isolated and enzymatically hydrolyzed to deoxyribonucleosides, prior to ultrafiltration and subsequent dilution to a sample solution consisting of water-acetonitrile-formic acid (98 : 2 : 0.2, v/v). The sample solution was loaded onto a 1 mm I.D. x 5 mm Hypercarb (5 mum) porous graphitic carbon trap column for analyte enrichment using an injection volume of 200 mul, and was subsequently back-flushed onto a 0.30 mm I.D. x 150 mm Lichrospher diol (5 mum) analytical column. The samples were loaded with a flow rate of 40 mul min(-1) and glyoxal-deoxyguanosine was desorbed from the trap column and eluted with an isocratic mobile phase consisting of water-acetonitrile-formic acid (50 : 50 : 0.2, v/v) at a flow rate of 5 mul min(-1). Mass spectrometric determination of glyoxal-deoxyguanosine was obtained by multiple reaction monitoring of the transition [M + H](+)m/z 326 --> m/z 210. The method was evaluated over the concentration range 0.25-50 ng ml(-1) of glyoxal-deoxyguanosine in the hydrolysate of 5 mug DNA. The method was linear with a correlation coefficient of 0.9998 in this range. The within-day (n = 6) and between-day (n = 6) precisions were determined as 1.2-11% and 1.4-11% RSD, respectively, and the recovery was close to 100%. The mass limit of detection was 15 pg, corresponding to a concentration limit of detection of 75 fg mul(-1) DNA hydrolysate solution, corresponding to 48 adducts per 10(6) normal nucleosides. The method was applied for the determination of glyoxal-deoxyguanosine in DNA hydrolysate solutions of calf thymus DNA and cell cultures after reaction or incubation with glyoxal.

Estuarine habitat quality reflects urbanization at large spatial scales in South Carolina's coastal zone

Land cover patterns were evaluated in 29 estuarine watersheds of South Carolina to determine relationships between urban/suburban development and estuarine habitat quality. Principal components analysis and Pearson product moment correlation analyses were used to examine the relationships between ten land cover categories and selected measures of nutrient or bacterial enrichment in the water column and contaminant enrichment in sediments. These analyses indicated strong relationships between land cover categories representing upland development and a composite measure of 24 inorganic and organic contaminants using the Effect Range Median-Quotient (ERM-Q). Similar relationships also were observed for the summed concentrations of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), pesticides, and metals. Data obtained from tidal creeks generally showed stronger correlations between urban/suburban land use and pesticides and metals compared to data obtained from larger open water habitats. Correlations between PAH concentrations and the urban/suburban land cover categories were similar between creek and open water habitats. PCB concentrations generally showed very little relationship to any of the land cover categories. Measures of nutrient enrichment, which included total Kjeldahl nitrogen (TKN), nitrate–nitrite, phosphorus, chlorophyll-a, and total organic carbon, were generally not significantly correlated with any land cover categories, whereas fecal coliform bacteria were significantly and positively correlated with the urban/suburban land cover categories and negatively correlated with the non-urban land cover categories. Fecal coliform correlations were stronger using data from the open water sites than from the tidal creek sites. Both ERM-Q and fecal coliform concentrations were much greater and more pervasive in watersheds with relatively high (>50%) urban/suburban cover compared to watersheds with low (<30%) urban/suburban cover. These analyses support the hypotheses that estuarine habitat quality reflects upland development patterns at large spatial scales, and that upland urbanization can result in increased risk of biological degradation and reduced safe human use of South Carolina's coastal resources.

A Metal-coded Affinity Tag Approach to Quantitative Proteomics

The quantitative analysis of protein mixtures is pivotal for the understanding of variations in the proteome of living systems. Therefore, approaches have been recently devised that generally allow the relative quantitative analysis of peptides and proteins. Here we present proof of concept of the new metal-coded affinity tag (MeCAT) technique, which allowed the quantitative determination of peptides and proteins. A macrocyclic metal chelate complex (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)) loaded with different lanthanides (metal(III) ions) was the essential part of the tag. The combination of DOTA with an affinity anchor for purification and a reactive group for reaction with amino acids constituted a reagent that allowed quantification of peptides and proteins in an absolute fashion. For the quantitative determination, the tagged peptides and proteins were analyzed using flow injection inductively coupled plasma MS, a technique that allowed detection of metals with high precision and low detection limits. The metal chelate complexes were attached to the cysteine residues, and the course of the labeling reaction was followed using SDS-PAGE and MALDI-TOF MS, ESI MS, and inductively coupled plasma MS. To limit the width in isotopic signal spread and to increase the sensitivity for ESI analysis, we used the monoisotopic lanthanide macrocycle complexes. Peptides tagged with the reagent loaded with different metals coelute in liquid chromatography. In first applications with proteins, the calculated detection limit for bovine serum albumin for example was 110 amol, and we have used MeCAT to analyze proteins of the Sus scrofa eye lens as a model system. These data showed that MeCAT allowed quantification not only of peptides but also of proteins in an absolute fashion at low concentrations and in complex mixtures.

Plasma cell purification from murine bone marrow using a two-step isolation approach

Interest in plasma cells has increased greatly in the past decade. While several studies have examined the longevity and transcriptional control of antibody secreting cells in vivo, few studies have examined freshly isolated plasma cells ex vivo. Studies of primary plasma cells have been limited primarily due to the difficulty of isolating the large numbers of plasma cells necessary for experiments. In this protocol, plasma cells are purified from murine bone marrow in approximately 5 hours, following a two-step isolation method utilizing column enrichment and FACS-sorting based on CD138 (Syndecan-1) surface expression.