Initial development and validation of a novel extraction method for quantitative mining of the formalin-fixed, paraffin-embedded tissue proteome for biomarker investigations

Annotated formalin-fixed, paraffin-embedded (FFPE) tissue archives constitute a valuable resource for retrospective biomarker discovery. However, proteomic exploration of archival tissue is impeded by extensive formalin-induced covalent cross-linking. Robust methodology enabling proteomic profiling of archival resources is urgently needed. Recent work is beginning to support the feasibility of biomarker discovery in archival tissues, but further developments in extraction methods which are compatible with quantitative approaches are urgently needed. We report a cost-effective extraction methodology permitting quantitative proteomic analyses of small amounts of FFPE tissue for biomarker investigation. This surfactant/heat-based approach results in effective and reproducible protein extraction in FFPE tissue blocks. In combination with a liquid chromatography-mass spectrometry-based label-free quantitative proteomics methodology, the protocol enables the robust representative and quantitative analyses of the archival proteome. Preliminary validation studies in renal cancer tissues have identified typically 250-300 proteins per 500 ng of tissue with 1D LC-MS/MS with comparable extraction in FFPE and fresh frozen tissue blocks and preservation of tumor/normal differential expression patterns (205 proteins, r = 0.682; p < 10(-15)). The initial methodology presented here provides a quantitative approach for assessing the potential suitability of the vast FFPE tissue archives as an alternate resource for biomarker discovery and will allow exploration of methods to increase depth of coverage and investigate the impact of preanalytical factors.     

The stability of the circulating human proteome to variations in sample collection and handling procedures measured with an aptamer-based proteomics array

Blood-based protein biomarkers hold great promise to advance medicine with applications that detect and diagnose diseases and aid in their treatment. We are developing such applications with our proteomics technology that combines high-content with low limits of detection. Biomarker discovery relies heavily on archived blood sample collections. Blood is dynamic and changes with different sampling procedures potentially confounding biomarker studies. In order to better understand the effects of sampling procedures on the circulating proteome, we studied three sample collection variables commonly encountered in archived sample sets. These variables included (1) three different sample tube types, PPT plasma, SST serum, and Red Top serum, (2) the time from venipuncture to centrifugation, and (3) the time from centrifugation to freezing. We profiled 498 proteins for each of 240 samples and compared the results by ANOVA. The results found no significant variation in the measurements for most proteins (~ 99%) when the two sample processing times tested were 2 h or less, regardless of sample tube type. Even at the longest timepoints, 20 h, ~ 82% of the proteins, on average for the three collection tube types, showed no significant change. These results are encouraging for proteomic biomarker discovery.     

In situ protein microarrays capable of real-time kinetics analysis based on surface plasmon resonance imaging

In recent years, in situ protein synthesis microarray technologies have enabled protein microarrays to be created on demand just before they are needed. In this paper, we utilized the TUS-TER immobilization technology to allow label-free detection with real-time kinetics of protein–protein interactions using surface plasmon resonance imaging (SPRi). We constructed an expression-ready plasmid DNA with a C-terminal TUS fusion tag to directionally immobilize the in situ synthesized recombinant proteins onto the surface of the biosensor. The expression plasmid was immobilized on the polyethylene imine-modified gold surface, which was then coupled with a cell-free expression system on the flow cell of the SPRi instrument. The expressed TUS fusion proteins bind on the surface via the immobilized TER DNA sequence with high affinity (∼3–7 × 10⁻¹³ M). The expression and immobilization of the recombinant in situ expressed proteins were confirmed by probing with specific antibodies. The present study shows a new low cost method for in situ protein expression microarrays that has the potential to study the kinetics of protein–protein interactions. These protein microarrays can be created on demand without the problems of stability associated with protein arrays used in the drug discovery and biomarker discovery fields.     

Toward a standardized urine proteome analysis methodology

Urine is an easily accessible bodily fluid particularly suited for the routine clinical analysis of disease biomarkers. Actually, the urinary proteome is more diverse than anticipated a decade ago. Hence, significant analytical and practical issues of urine proteomics such as sample collection and preparation have emerged, in particular for large-scale studies. We have undertaken a systematic study to define standardized and integrated analytical protocols for a biomarker development pipeline, employing two LC-MS analytical platforms, namely accurate mass and time tags and selected reaction monitoring, for the discovery and verification phase, respectively. Urine samples collected from hospital patients were processed using four different protocols, which were evaluated and compared on both analytical platforms. Addition of internal standards at various stages of sample processing allowed the estimation of protein extraction yields and the absolute quantification of selected urinary proteins. Reproducibility of the entire process and dynamic range of quantification were also evaluated. Organic solvent precipitation followed by in-solution digestion provided the best performances and was thus selected as the standard method common to the discovery and verification phases. Finally, we applied this protocol for platforms' cross-validation and obtained excellent consistency between urinary protein concentration estimates by both analytical methods performed in parallel in two laboratories.     

Discrepancy between radioimmunoassay and high performance liquid chromatography tandem-mass spectrometry for the analysis of androstenedione

The discrepancy of results for the quantification of androstenedione in human serum between a radioimmunoassay (RIA) method and high performance liquid chromatography tandem-mass spectrometry (LC–MS/MS) was investigated. RIA overestimated concentrations compared to LC–MS/MS on 59 clinical samples (RIA = 1.79 × LC–MS/MS + 0.94). RIA kit and LC–MS/MS calibrants were also determined by both methods. The RIA performed with improved accuracy on the calibrants (RIA = 1.35 × LC–MS/MS − 0.28). Lipid, protein, electrolyte content, and pH of the two sets of calibrants were further investigated. The RIA calibrants contained little lipid material, while the LC–MS/MS calibrant material contained the same levels expected in normal serum/plasma. The pH and sex hormone binding globulin (SHBG) values were different between the RIA calibrants and the LC–MS/MS calibrant material (SHBG, 31 ± 2 and 38 ± 2 nmol/l; pH, 8.27 ± 0.18 and 8.66 ± 0.03, respectively). No correlation was observed between androstenedione RIA and LC–MS/MS discrepancy and lipid or protein. LC–MS/MS sample preparation was tested for the removal of protein-bound material and recovery determined (99–108%). The corresponding RIA results overestimated androstenedione by 52–174% compared to LC–MS/MS. The results here demonstrate that LC–MS/MS is the more accurate method.     

Progressive degradation of serum samples limits proteomic biomarker discovery

Preanalytical variables play a key role in discovery of biomarkers. Although the effect of several preanalytical variables on the mass spectral profiles has been studied extensively, little is known about long-term storage of serum samples. This is important because samples used in case-control or epidemiological studies are usually stored for a long time before analysis. Here we evaluated long-term storage effects on mass spectral peak patterns of serum peptides extracted using weak cation exchange magnetic beads. For this, 20 serum samples stored at −80 °C were divided equally into two groups based on their storage time. We found that intensities of 26 mass spectral peaks significantly varied between these two groups. Intensities of these peaks significantly correlated with storage time. Genetic algorithm-based models generated using these 26 peaks could classify 63 additional samples into these two groups with 100% and 96% accuracy, respectively. We also show that storing samples for 10 months at −80 and −20 °C results in the appearance/disappearance or intensity variation of peaks, some of which were previously reported as disease biomarkers.     

Evaluation of multiprotein immunoaffinity subtraction for plasma proteomics and candidate biomarker discovery using mass spectrometry

Strategies for removal of high abundance proteins have been increasingly utilized in proteomic studies of serum/plasma and other body fluids to enhance the detection of low abundance proteins and achieve broader proteome coverage; however, both the reproducibility and specificity of the high abundance protein depletion process still represent common concerns. Here we report a detailed evaluation of immunoaffinity subtraction performed applying the ProteomeLab IgY-12 system that is commonly used in human serum/plasma proteome characterization in combination with high resolution LC-MS/MS. Plasma samples were repeatedly processed using this approach, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. The removal of target proteins by the immunoaffinity subtraction system and the overall process was highly reproducible. Non-target proteins, including one spiked protein standard (rabbit glyceraldehyde-3-phosphate dehydrogenase), were also observed to bind to the column at different levels but also in a reproducible manner. The results suggest that multiprotein immunoaffinity subtraction systems can be readily integrated into quantitative strategies to enhance detection of low abundance proteins in biomarker discovery studies.     

Development and validation of a Sensitive bioanalytical method for the quantitative estimation of Pantoprazole in human plasma samples by LC–MS/MS: Application to bioequivalence study

The present study aims at developing a simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantification of pantoprazole sodium (PS) in human plasma using pantoprazole D3 (PSD3) as internal standard (IS). Chromatographic separation was performed on Zorbax SB-C18, 4.6 mm × 75 mm, 3.5 μm, 80 Å column with an isocratic mobile phase composed of 10 mM ammonium acetate (pH 7.10): acetonitrile (30:70, v/v), pumped at 0.6 mL/min. PS and PSD3 were detected with proton adducts at m/z 384.2 → 200.1 and 387.1 → 203.1 in multiple reaction monitoring (MRM) positive mode, respectively. Precipitation method was employed in the extraction of PS and PSD3 from the biological matrix. This method was validated over a linear concentration range of 10.00–3000.00 ng/mL with correlation coefficient (r) ≥ 0.9997. Intra- and inter-day precision of PS were found to be within the range of 1.13–1.54 and 1.76–2.86, respectively. Both analytes were stable throughout freeze/thaw cycles, bench top and postoperative stability studies. This method was successfully utilized in the analysis of blood samples following oral administration of PS (40 mg) in healthy human volunteers.     

Microextraction in packed sorbent for analysis of antidepressants in human plasma by liquid chromatography and spectrophotometric detection

A sensitive and reproducible method by microextraction packed sorbent and liquid chromatography with UV detection (MEPS/LC–UV) is described for the determination of new generation antidepressants (sertraline, mirtazapine, fluoxetine, citalopram and paroxetine) in human plasma samples. The MEPS variables, such as sample volume, pH, number of extraction cycles (draw–eject), and desorption conditions (solvent and solvent volume of elution) influenced the MEPS/LC efficiency significantly. Important factors in the optimization of MEPS efficiency, as well as washing steps and carryover effect are discussed. The analyses were carried out using small sample volumes (400 μL), and in a short time period (3 min for the entire sample preparation step). The MEPS/LC–UV method was shown to be linear at concentrations ranging from the limit of quantification (LOQ) to 1000 ng mL⁻¹. The LOQ values ranged from 10 to 25 ng mL⁻¹. The inter-day precision of the method presented coefficient of the variation ranging from 1.3% to 8.7%. On the basis of analytical validation, it is shown that the MEPS/LC–UV methodology is adequate for antidepressant analysis, from therapeutic to toxic levels. In order to evaluate the proposed method for clinical use, the MEPS/LC–UV method was applied to analysis of plasma samples from elderly depressed patients.     

Proteomic analysis of eccrine sweat: implications for the discovery of schizophrenia biomarker proteins

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and multiple reaction monitoring mass spectrometry (MRM-MS) proteomics analyses were performed on eccrine sweat of healthy controls, and the results were compared with those from individuals diagnosed with schizophrenia (SZ). This is the first large scale study of the sweat proteome. First, we performed LC-MS/MS on pooled SZ samples and pooled control samples for global proteomics analysis. Results revealed a high abundance of diverse proteins and peptides in eccrine sweat. Most of the proteins identified from sweat samples were found to be different than the most abundant proteins from serum, which indicates that eccrine sweat is not simply a plasma transudate and may thereby be a source of unique disease-associated biomolecules. A second independent set of patient and control sweat samples were analyzed by LC-MS/MS and spectral counting to determine qualitative protein differential abundances between the control and disease groups. Differential abundances of selected proteins, initially determined by spectral counting, were verified by MRM-MS analyses. Seventeen proteins showed a differential abundance of approximately 2-fold or greater between the SZ pooled sample and the control pooled sample. This study demonstrates the utility of LC-MS/MS and MRM-MS as a viable strategy for the discovery and verification of potential sweat protein disease biomarkers.     

Comparative proteome analyses of human plasma following in vivo lipopolysaccharide administration using multidimensional separations coupled with tandem mass spectrometry

There is significant interest in characterization of the human plasma proteome due to its potential for providing biomarkers applicable to clinical diagnosis and treatment and for gaining a better understanding of human diseases. We describe here a strategy for comparative proteome analyses of human plasma, which is applicable to biomarker identifications for various disease states. Multidimensional liquid chromatography-mass spectrometry (LC-MS/MS) has been applied to make comparative proteome analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Peptide peak areas and the number of peptide identifications for each protein were used to evaluate the reproducibility of LC-MS/MS and to compare relative changes in protein concentration between the samples following LPS treatment. A total of 804 distinct plasma proteins (not including immunoglobulins) were confidently identified with 32 proteins observed to be significantly increased in concentration following LPS administration, including several known inflammatory response or acute-phase mediators such as C-reactive protein, serum amyloid A and A2, LPS-binding protein, LPS-responsive and beige-like anchor protein, hepatocyte growth factor activator, and von Willebrand factor, and thus, constituting potential biomarkers for inflammatory response.     

Global stability of plasma proteomes for mass spectrometry-based analyses

Peptide-based mass spectrometry approaches, such as multiple reaction monitoring, provide a powerful means to measure candidate protein biomarkers in plasma. A potential confounding problem is the effect of preanalytical variables, which may affect the integrity of proteins and peptides. Although some blood proteins undergo rapid physiological proteolysis ex vivo, the stability of most plasma proteins to preanalytical variables remains largely unexplored. We applied liquid chromatography-tandem mass spectrometry shotgun proteomics and multiple reaction monitoring analyses to characterize the stability of proteins at the peptide level in plasma. We systematically evaluated the effects of delay in plasma preparation at different temperatures, multiple freeze-thaw cycles and erythocyte hemolysis on peptide and protein inventories in prospectively collected human plasma. Time course studies indicated few significant changes in peptide and protein identifications, semitryptic peptides and methionine-oxidized peptides in plasma from blood collected in EDTA plasma tubes and stored for up to a week at 4 °C or room temperature prior to plasma isolation. Similarly, few significant changes were observed in similar analyses of plasma subjected to up to 25 freeze-thaw cycles. Hemolyzed samples produced no significant differences beyond the presence of hemoglobin proteins. Finally, paired comparisons of plasma and serum samples prepared from the same patients also yielded few significant differences, except for the depletion of fibrinogen in serum. Blood proteins thus are broadly stable to preanalytical variables when analyzed at the peptide level. Collection protocols to generate plasma for multiple reaction monitoring-based analyses may have different requirements than for other analyses directed at intact proteins.     

Low abundance protein enrichment for discovery of candidate plasma protein biomarkers for early detection of breast cancer

Molecular biomarkers of early stage breast cancer may improve the sensitivity and specificity of diagnosis. Plasma biomarkers have additional value in that they can be monitored with minimal invasiveness. Plasma biomarker discovery by genome-wide proteomic methods is impeded by the wide dynamic range of protein abundance and the heterogeneity of protein expression in healthy and disease populations which requires the analysis of a large number of samples. We addressed these issues through the development of a novel protocol that couples a combinatorial peptide ligand library protein enrichment strategy with isobaric label-based 2D LC-MS/MS for the identification of candidate biomarkers in high throughput. Plasma was collected from patients with stage I breast cancer or benign breast lesions. Low abundance proteins were enriched using a bead-based combinatorial library of hexapeptides. This resulted in the identification of 397 proteins, 22% of which are novel plasma proteins. Twenty-three differentially expressed plasma proteins were identified, demonstrating the effectiveness of the described protocol and defining a set of candidate biomarkers to be validated in independent samples. This work can be used as the basis for the design of properly powered investigations of plasma protein expression for biomarker discovery in larger cohorts of patients with complex disease.     

Strategy combining separation of isotope-labeled unfolded proteins and matrix-assisted laser desorption/ionization mass spectrometry analysis enables quantification of a wide range of serum proteins

A novel strategy for the quantitative profiling of serum proteome is described. It includes an ammonium sulfate depletion of the serum, an affordable stable isotope labeling chemistry for samples with a large amount of protein, separation of the unfolded proteins, and relative quantification by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Labeling of unfolded proteins was performed using normal (D₀) acrylamide and deuterated (D₃) acrylamide. The workflow for separating the unfolded proteins includes whole gel elution and ion exchange liquid chromatography, and it combines electrophoretic separation based on the protein molecular weight followed by chromatographic separation in the presence of 8 M urea based on protein charge. This was followed by trypsinolysis and MALDI MS analysis, leading to the quantification of a large number of serum proteins, including those with an abundance of 10^-5 less than albumin. This robust and inexpensive workflow is suitable for the quantitative profiling of protein changes in serum associated with preanalytical variables.     

Enhanced detection of low abundance human plasma proteins using a tandem IgY12-SuperMix immunoaffinity separation strategy

The enormous dynamic range of human bodily fluid proteomes poses a significant challenge for current MS-based proteomics technologies as it makes it especially difficult to detect low abundance proteins in human biofluids such as blood plasma, which is an essential aspect for successful biomarker discovery efforts. Here we present a novel tandem IgY12-SuperMix immunoaffinity separation system for enhanced detection of low abundance proteins in human plasma. The tandem IgY12-SuperMix system separates approximately 60 abundant proteins from the low abundance proteins in plasma, allowing for significant enrichment of low abundance plasma proteins in the SuperMix flow-through fraction. High reproducibility of the tandem separations was observed in terms of both sample processing recovery and LC-MS/MS identification results based on spectral count data. The ability to quantitatively measure differential protein abundances following application of the tandem separations was demonstrated by spiking six non-human standard proteins at three different levels into plasma. A side-by-side comparison between the SuperMix flow-through and IgY12 flow-through samples analyzed by both one- and two-dimensional LC-MS/MS revealed a 60-80% increase in proteome coverage as a result of the SuperMix separations, suggesting significantly enhanced detection of low abundance proteins. A total of 695 plasma proteins were confidently identified in a single analysis (with a minimum of two peptides per protein) by coupling the tandem separation strategy with two-dimensional LC-MS/MS, including 42 proteins with reported normal concentrations of approximately 100 pg/ml to 100 ng/ml. The concentrations of two selected proteins, macrophage colony-stimulating factor 1 and matrix metalloproteinase-8, were independently validated by ELISA as 202 pg/ml and 12.4 ng/ml, respectively. Evaluation of binding efficiency revealed that 45 medium abundance proteins were efficiently captured by the SuperMix column with >90% retention. Taken together, these results illustrate the potential broad utilities of this tandem IgY12-SuperMix strategy for proteomics applications involving human biofluids where effectively addressing the dynamic range challenge of the specimen is imperative.     

Integrated pipeline for mass spectrometry-based discovery and confirmation of biomarkers demonstrated in a mouse model of breast cancer

Despite their potential to impact diagnosis and treatment of cancer, few protein biomarkers are in clinical use. Biomarker discovery is plagued with difficulties ranging from technological (inability to globally interrogate proteomes) to biological (genetic and environmental differences among patients and their tumors). We urgently need paradigms for biomarker discovery. To minimize biological variation and facilitate testing of proteomic approaches, we employed a mouse model of breast cancer. Specifically, we performed LC-MS/MS of tumor and normal mammary tissue from a conditional HER2/Neu-driven mouse model of breast cancer, identifying 6758 peptides representing >700 proteins. We developed a novel statistical approach (SASPECT) for prioritizing proteins differentially represented in LC-MS/MS datasets and identified proteins over- or under-represented in tumors. Using a combination of antibody-based approaches and multiple reaction monitoring-mass spectrometry (MRM-MS), we confirmed the overproduction of multiple proteins at the tissue level, identified fibulin-2 as a plasma biomarker, and extensively characterized osteopontin as a plasma biomarker capable of early disease detection in the mouse. Our results show that a staged pipeline employing shotgun-based comparative proteomics for biomarker discovery and multiple reaction monitoring for confirmation of biomarker candidates is capable of finding novel tissue and plasma biomarkers in a mouse model of breast cancer. Furthermore, the approach can be extended to find biomarkers relevant to human disease.     

LC–MS/MS method development and validation for the determination of polymyxins and vancomycin in rat plasma

Simple, sensitive and robust liquid chromatography–tandem mass spectrometer (LC–MS/MS) methods were developed and validated for the determination of lipopeptide polymyxins and glycopeptide vancomycin in rat plasma. The effect of trichloroacetic acid (TCA) concentration on sample recoveries (peak area of sample recovered from plasma/peak area of sample from neat solvent solutions) was studied and an optimized concentration of 30% TCA were determined that gives the best sample recovery for the peptides from rat plasma. The effect of the TCA concentration on the chromatographic behavior of peptides was studied on a Phenomenex Jupiter C18 5 μ 300 Å 50 mm × 2 mm column using a mobile phase with a pH of 2.8. Other than protein precipitation, TCA also acted as ion pairing reagent and was only present in the samples but not in the mobile phases. The data demonstrated that by increasing the TCA concentration, the analyte retention and sensitivity were improved. The absence of TCA in mobile phase helped to reduce the ion source contamination and to achieve good reproducibility. The plasma method was linearly calibrated from 5 to 5000 ng/mL for polymyxins with precisions to be of 2.3–10.8%, and accuracies to be 91.7–107.4% for polymyxin B1, B2, E1, E2, respectively. For vancomycin the calibration is from 1 to 5000 ng/mL with precisions to be of 7.8–10.3 and accuracies to be 96.2–102.0%. The LLOQs corresponding with a coefficient of variation less than 20% were 7.5, 18.1, 7.3, 5.0 and 1.0 ng/mL for polymyxin B1, B2, E1, E2 and vancomycin, respectively.     

mTRAQ-based quantitative analysis combined with peptide fractionation based on cysteinyl peptide enrichment

In the present study, the fractionation scheme for cysteinyl peptide enrichment (CPE) was combined with the mass differential tags for relative and absolute quantification (mTRAQ) method to reduce sample complexity and increase proteome coverage. Cysteine residues of the proteins were first alkylated using iodoacetyl PEG₂–biotin instead of other conventional alkylating agents such as iodoacetamide. After trypsin digestion, amine groups were labeled with mTRAQ, and these labeled peptides were fractionated according to the presence or absence of cysteine residues using avidin–biotin affinity chromatography. With these approaches, we were able to divide the peptides into the two fractions with more than 90% fractionation efficiency for standard protein and MCF7 cell lysate. When the fractionation strategy was applied to colorectal cancer tissue samples, we were able to obtain quantitative information that was consistent with the previous study based on mTRAQ quantification, implying that the cysteine-based fractionation method does not affect mTRAQ quantification. We expect that the mTRAQ-based quantitative analysis combined with peptide fractionation through the CPE strategy would allow for deep-down analysis of proteome samples and ultimately for increasing proteome coverage with simultaneous quantification for biomarker discovery.     

Combined effects of temperature and cadmium exposure on haemocyte apoptosis and cadmium accumulation in the eastern oyster Crassostrea virginica (Gmelin)

Combined effects of acclimation temperature (12, 20 and 28 °C) and exposure to a toxic metal cadmium (Cd, 50 μg L⁻¹) on haemolymph parameters related to immune defense and metal transport were studied in a model marine bivalve, Crassostrea virginica. Acclimation to elevated temperatures resulted in higher plasma protein concentrations and increased Cd levels in oyster haemolymph plasma and haemocytes. Cd accumulation in haemocytes was linear over the 45 days of Cd exposure and accumulation rates were 0.10, 0.53 and 0.56 μg Cd g⁻¹ dry mass at 12, 20 and 28 °C, respectively. Percentage of blood Cd burden associated with haemocytes increased with increasing temperatures from 13–20% at 12 °C to 26–47% at 20 and 28 °C suggesting a higher role for cellular Cd transport at elevated temperatures. Cd levels in gills and hepatopancreas were positively correlated with Cd concentration in haemocytes, but accumulation rates were considerably faster, so that after 45 days of exposure Cd levels in gills and hepatopancreas were >10–20 times higher than in haemocytes. As a result of slow Cd accumulation possibly reflecting fast haemocyte turnover rates and/or exocytosis of Cd-containing granules, haemocytes in Cd-exposed oysters did not reach threshold Cd burdens required to trigger apoptosis. This suggests that haemocyte viability is not likely to contribute to immunosuppression in the environmentally relevant Cd range. In contrast, elevated temperature (28 °C) resulted in a significant increase in the percentage of apoptotic haemocytes compared to 12 or 20 °C supporting the notion that 28 °C is physiologically stressful for C. virginica. Overall, our study demonstrates strong effects of environmental temperature on haemocyte viability and other important blood parameters such as plasma protein content and metal transport capability which may mask potential Cd effects at environmentally relevant exposure levels.     

Identification and quantitation of human amniotic fluid components using capillary zone electrophoresis

A capillary electrophoresis method was used to measure albumin, immunoglobulin G (IgG), transferrin, and uric acid in 230 amniotic fluid (AF) samples collected at 15.15 ± 0.06 weeks gestation. Species were quantified by external calibration using thiamine as internal standard. All major components were detected within 10 min. Migration time reproducibility was 3.0% relative standard deviation (RSD) and normalized peak areas were 12% RSD or better at 190 nm from 81 measurements of a pooled AF sample. The separation profile was not affected by 10 h of storage at room temperature or by 10 freeze-thaw cycles, suggesting that frozen AF samples are suitable for protein biomarker studies.