Reproducible Profiling Research Articles

Sensitive and Reproducible Intact Mass Analysis of Complex Protein Mixtures with Superficially Porous Capillary Reversed-Phase Liquid Chromatography Mass Spectrometry

The compatibility of superficially porous (SP) resin for label-free intact protein analysis with online capillary LC/MS is demonstrated to give improved chromatographic resolution, sensitivity, and reproducibility. The robustness of the platform was measured against several samples of varying complexity and sample loading amount. The results indicate that capillary SP columns provide high loading capacities and that ∼6 s chromatographic peak widths are typical for standard proteins in simple mixtures and proteins isolated from cell and tissue lysates. Subfemtomole detection limits for standard proteins were consistently observed, with the lowest levels at 12 amol for ubiquitin. The analysis of total heart homogenates shows that capillary SP columns provide theoretical peak capacity of 106 protein forms with 30 min total analysis time and enabled detection of proteins from complex mixtures with a single high-resolution scan. The SPLC/MS platform also detected 343 protein forms from two HeLa acid extract replicate analyses that consumed 5 × 10(4) cells and 30 min analysis time, each. Comparison of all the species observed in each HeLa replicate showed 90% overlap (309 forms) with a Pearson correlation coefficient of 89.9% for the common forms observed in the replicates. Efficient acid extract of 1 × 10(4) HeLa cells allowed reproducible detection of common modification states and members from all five of the histone families and demonstrated that capillary SPLC/MS supports reproducible label-free profiling of histones in <15 min total analysis time. The data presented demonstrate that a capillary LC/MS platform utilizing superficially porous stationary phase and a LTQ-Orbitrap FT-MS is fast, sensitive, and reproducible for intact protein profiling from small tissue and cell amounts.

Preanalytical standardization of amino acid and acylcarnitine metabolite profiling in human blood using tandem mass spectrometry

Quantitative metabolite profiling in biological samples has the potential to reflect physiological status and to identify disease associated disturbances in metabolic networks. However, this approach is hampered by a wide range of preanalytical variables. Hence, the aim of our study was to develop a standardized preanalytical protocol for metabolite profiling of amino acids and acylcarnitines in human blood. Amino acids and acylcarnitines were simultaneous analyzed after butylation of 3 μL dried blood or 10 μL whole blood, serum and anticoagulated plasma using electrospray tandem-mass spectrometry. The influence of exogenous and endogenous preanalytical variables was investigated in healthy volunteers. Different sampling materials and anticoagulants for blood taking were investigated. Concentrations of long-chain acylcarnitines were 5-fold higher in EDTA-whole blood or dried whole blood compared to serum and anticoagulated plasma. Significant differences in amino acid concentrations were found for capillary versus venous blood taking. Fasting for 8 h before specimen collection minimized the nutritional influence. Physical activity significantly alters amino acid and short chain acylcarnitine concentrations. As a result of our preanalytical investigation we developed a pre-treatment protocol based on EDTA whole blood dried on filter paper to reduce the preanalytical variability and facilitate reproducible quantitative metabolite profiling in clinical trials.

Finding diabetic nephropathy biomarkers in the plasma peptidome by high‐throughput magnetic bead processing and MALDI‐TOF‐MS analysis

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease and improved biomarkers would help identify high-risk individuals. The aim of this study was to discover candidate biomarkers for DN in the plasma peptidome in an in-house cross-sectional cohort (n=122) of type 1 diabetic patients diagnosed with normo-, micro-, and macroalbuminuria. Automated, high-throughput, and reproducible (interassay median CV: 13-14%) plasma peptide profiling protocols involving RPC18 and weak cation exchange magnetic beads on a liquid handling workstation with a MALDI-TOF-MS readout were successfully established. Using these protocols and a combined univariate (Kruskal-Wallis) and multivariate (independent component analysis) statistical analysis approach, ten single peptides and three multi-peptide candidate biomarkers were found. Employment of RPC18 and weak cation exchange magnetic beads proved to be complementary. The proteins found in this study, including C3f and apolipoprotein C-I, represent new candidate biomarkers for DN from the plasma peptidome. The automated procedures and implementation of independent components analysis provide a fast and informative system for analyzing individual patient samples in protein biomarker discovery.

Standardized peptidome profiling of human cerebrospinal fluid by magnetic bead separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Peptidome profiling of human cerebrospinal fluid (CSF) is a promising tool to identify novel disease-associated biomarkers. Our aim was to develop a standardized protocol for reproducible peptidome profiling of CSF using magnetic bead (MB) separation followed by MALDI-TOF MS. Peptidome fractionation and profiling of CSF were performed using MBs with different surface functionalities. We investigated exogenous variables (storage conditions, freeze-thaw-cycles) and endogenous interferences (albumin, immunoglobulin, blood, leukocytes) in pooled CSF samples. We detected approximately 500 signals with an S/N ratio >10 and an overlap frequency of about 40% in non-pathological CSF. Within- and between-day imprecisions in relative signal intensities ranged from 3 to 28% and 7 to 47%, respectively. CSF storage at room temperature for up to 6 h and at 4 degrees C for up to 3 days did not significantly influence the mass spectra. Consecutive freeze-thaw-cycles significantly affected the mass spectra. High albumin and immunoglobulin content altered the CSF preparation using MB-HIC C8 beads. Blood contamination showed no effect on mass spectra up to a hemoglobin concentration of 0.075 micromol/L. The presence of leukocytes up to a cell number of 30 Mpt/L did not affect mass spectra. Our reliable pretreatment protocol allows standardization of preanalytical modalities and thereby enables reproducible peptidome profiling of human CSF using MB separation followed by MALDI-TOF MS.

MicroRNA expression profiling using microarrays

Microarray technology is a powerful high-throughput tool capable of monitoring the expression of thousands of small noncoding RNAs at once within tens of samples processed in parallel in a single experiment. To conduct a genome-wide analysis of miRNA expression of normal and disease samples, such as cancer, and to distinguish expression signatures associated with diagnosis, prognosis and therapeutic interventions, we have developed a unique miRNA microarray assay on a CodeLink platform. The miRNA array consists of 4,104 probes printed in duplicate. This array can simultaneously profile more than 1,500 mature miRNAs and their corresponding precursors from 474 human and 373 mouse miRNA genes. The full protocol details of the miRNA microarray assay developed by our group are described here, including miRNA oligo probe design, array fabrication and miRNA target preparation (by reverse transcription of total RNA), target-probe hybridization on array, signal detection and data analysis. The assay is simple, can be easily standardized and allows the reproducible profiling of up to 24 total RNA samples within 24 h.

In vivo alternative testing with zebrafish in ecotoxicology

Although rodents have previously been used in ecotoxicological studies, they are expensive, time-consuming, and are limited by strict legal restrictions. The present study used a zebrafish (Danio rerio) model and generated data that was useful for extrapolating toxicant effects in this system to that of humans. Here we treated embryos of the naive-type as well as a transiently transfected zebrafish liver cell line carrying a plasmid (phAhRE-EGFP), for comparing toxicity levels with the well-known aryl hydrocarbon receptor (AhR)-binding toxicants: 3,3',4,4',5-pentachlorobiphenyl (PCB126), 2,3,7,8-tetrachlorodibenzo-p-dioxin, and 3-methylcholanthrene. These toxicants induced a concentration-dependent increase in morphological disruption, indicating toxicity at early life-stages. The transient transgenic zebrafish liver cell line was sensitive enough to these toxicants to express the CYP1A1 regulated enhanced green fluorescent protein. The findings of this study demonstrated that the zebrafish in vivo model might allow for extremely rapid and reproducible toxicological profiling of early life-stage embryo development. We have also shown that the transient transgenic zebrafish liver cell line can be used for research on AhR mechanism studies.

Automated Sorting of Monocytes from Whole Blood for Reproducible Gene Expression Profiling.

Microarray-based gene expression profiling has been used to develop clinically relevant molecular classifiers for many different diseases. Furthermore, it has been shown for various chronic diseases that specific gene expression patterns are reflected at the level of blood cells. However, blood is a complex tissue comprising numerous cell types. Therefore, the contribution of rare cell types to a whole blood expression profile might not be detected and a substantial proportion of what is usually reported as “up-regulation” or “down-regulation” might actually be the result of a shift in cell populations and not of a true regulatory process. In order to circumvent these problems, several techniques have been established to analyze purified subpopulations rather than whole blood samples. Previously, it has been shown, for example, that reproducible gene expression profiles can be generated by positive selection of blood cell subsets from PBMCs1. As the preparation of PBMCs by, for example, Ficoll is time-consuming, inconvenient, and not amenable to automation, we have set up a combined direct whole blood cell separation and gene expression profiling protocol. By using Whole Blood CD14 MicroBeads in combination with the autoMACS Pro™ Separator, the separation protocol generally allowed enrichment of monocytes from whole blood within 30 min with purities higher than 90%. In combination with the depletion of neutrophils, the major source of contaminating RNA, purities increased to over 95% for all tested blood donors. Monocytes included the CD14bright/CD16− as well as the CD14dim/CD16+ populations. To assess the reproducibility of gene expression profiles and the influence of several experimental parameters, monocytes were sorted from 5 ml whole blood. RNA was extracted and hybridized to microarrays and the Pearson correlation coefficients of pairwise comparisons were calculated. Technical repeats of monocyte analysis from blood donated at different days showed a higher correlation coefficient than whole blood RNA. Blood storage at room temperature resulted in a strong deregulation of many genes, whereas blood stored at 4°C showed minimal changes, which is in agreement with previous studies. Skipping the centrifugation step, which is used to remove unbound MicroBeads did not alter the gene expression profiles. Incubation of sorted cells in PrepProtect™ Stabilization Buffer showed no alteration of gene expression thus enabling the shipping of cells without liquid nitrogen. Monocytes play a crucial role in diseases like atherosclerosis. Our rapid and simple protocol for combined direct cell sorting from whole blood and gene expression profiling of monocytes might help to ease the discovery of new biomarkers and to screen and monitor patients. 1 Lyons et al., BMC Genomics (2007), 8:64.

Comprehensive aneuploidy screening in single cells using microarray comparative genomic hybridization methods implications for preimplantation genetic diagnosis

To develop clinically applicable DNA microarray methods for comprehensive identification of genomic imbalances (ie aneuploidy) in isolated cells. Array comparative genomic hybridization (array CGH) together with whole genome amplification (WGA) were used to screen for copy number aberrations in single fibroblast cells derived from various aneuploid cell lines. DNA was extracted from single fibroblasts and amplified using degenerate oligonucleotide primed PCR (DOP-PCR). Test and reference DNA were labeled with Cy5 and Cy3 fluorescent dyes, respectively. The labeled DNAs were co-precipitated and hybridized to bacterial artificial chromosome (BAC)-arrays spotted with clones specific for every chromosome at an average resolution of 0.5 megabases. The array was then scanned and Cy5/Cy3 intensity ratios calculated. Ratio values close to 1 indicate a balanced chromosomal status whereas a ratio >1 or <1 denotes a corresponding gain or a loss of genetic material in the test sample. DOP-PCR mediated DNA amplification and array CGH permitted reproducible genomic profiling of single cells. Trisomies of chromosomes 13, 15, 16, 18, and 21 along with balanced ploidy levels of the remaining chromosomes were identified in single aneuploid fibroblast cells. Sex mismatched reference DNA was also accurately determined. Conventional PGD techniques, which rely on fluorescence in situ hybridization (FISH) analysis, usually screen <1/2 of the human chromosomes and consequently many oocytes or embryos carrying lethal abnormalities are not identified. Array CGH together with whole genome amplification allows comprehensive aneuploidy testing and detection of unbalanced translocations in single cells in a manner compatible with transfer during an IVF cycle. Sufficient DNA is also available following amplification to permit testing for single gene defects. This diagnostic may lead to improved IVF outcomes as all aneuploidies can be avoided and could potentially result in a reduction of the multiple birth rate as pregnancy rates could be maintained with fewer embryos transferred.

Standardized Peptidome Profiling of Human Urine by Magnetic Bead Separation and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Peptidome profiling of human urine is a promising tool to identify novel disease-associated biomarkers; however, a wide range of preanalytical variables influence the results of peptidome analysis. Our aim was to develop a standardized protocol for reproducible urine peptidome profiling by means of magnetic bead (MB) separation followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). MBs with defined surface functionalities (hydrophobic interaction, cation exchange, and metal ion affinity) were used for peptide fractionation of urine. Mass accuracy and imprecision were calculated for 9 characteristic mass signals (M(r), 1000-10,000). Exogenous variables (instrument performance, urine sampling/storage conditions, freezing conditions, and freeze-thaw cycles) and endogenous variables (pH, urine salt and protein concentrations, and blood and bacteria interferences) were investigated with urine samples from 10 male and 10 female volunteers. We detected 427 different mass signals in the urine of healthy donors. Within- and between-day imprecision in relative signal intensities ranged from 1% to 14% and from 4% to 16%, respectively. Weak cation-exchange and metal ion affinity MB preparations required adjustment of the urinary pH to 7. Storage time, storage temperature, the number of freeze-thaw cycles, and bacterial and blood contamination significantly influenced urine peptide patterns. Individual urine peptide patterns differed significantly within and between days. This imprecision was diminished by normalization to a urinary protein content of 3.5 microg. This reliable pretreatment protocol allows standardization of preanalytical modalities and facilitates reproducible peptidome profiling of human urine by means of MB separation in combination with MALDI-TOF MS.

SuperSAGE array: the direct use of 26-base-pair transcript tags in oligonucleotide arrays

We developed a new platform for genome-wide gene expression analysis in any eukaryotic organism, which we called SuperSAGE array. The SuperSAGE array is a microarray onto which 26-bp oligonucleotides corresponding to SuperSAGE tag sequences are directly synthesized. A SuperSAGE array combines the advantages of the highly quantitative SuperSAGE expression analysis with the high-throughput microarray technology. We demonstrated highly reproducible gene expression profiling by the SuperSAGE array for 1,000 genes (tags) in rice. We also applied this technology to the detailed study of expressed genes identified by SuperSAGE in Nicotiana benthamiana, an organism for which sufficient genome sequence information is not available. We propose that the SuperSAGE array system represents a new paradigm for microarray construction, as no genomic or cDNA sequence data are required for its preparation.

Qdot Nanobarcodes for Multiplexed Gene Expression Analysis

We report a quantum dot (Qdot) nanobarcode-based microbead random array platform for accurate and reproducible gene expression profiling in a high-throughput and multiplexed format. Four different sizes of Qdots, with emissions at 525, 545, 565, and 585 nm are mixed with a polymer and coated onto the 8-mum-diameter magnetic microbeads to generate a nanobarcoded bead termed as QBeads. Twelve intensity levels for each of the four colors were used. Gene-specific oligonucleotide probes are conjugated to the surface of each spectrally nanobarcoded bead to create a multiplexed panel, and biotinylated cRNAs are generated from sample total RNA and hybridized to the gene probes on the microbeads. A fifth streptavidin Qdot (655 nm or infrared Qdot) binds to biotin on the cRNA, acting as a quantification reporter. Target identity was decoded based on spectral profile and intensity ratios of the four coding Qdots (525, 545, 565, and 585 nm). The intensity of the 655 nm Qdot reflects the level of biotinylated cRNA captured on the beads and provides the quantification for the corresponding target gene. The system shows a sensitivity of < or =10(4) target molecules detectable with T7 amplification, a level that is better than the 10(5) number achievable with a high-density microarray system, and approaching the 10(3)-10(4) level usually observed for quantitative PCR (qPCR). The QBead nanobarcode system has a dynamic range of 3.5 logs, better than the 2-3 logs observed on various microarray platforms. The hybridization reaction is performed in liquid phase and completed in 1-2 hours, at least 1 order of magnitude faster than microarray-based hybridizations. Detectable fold change is lower than 1.4-fold, showing high precision even at close to single copy per cell level. Reproducibility for this proof-of-concept study approaches that of Affymetrix GeneChip microarray, with an R(2) value between two repeats at 0.984, and interwell CV around 5%. In addition, it provides increased flexibility, convenience, and cost-effectiveness in comparison to conventional gene expression profiling methods.

Gene expression profiling in formalin-fixed, paraffin-embedded (FFPE) benign and cancerous prostate tissues using universal bead arrays

9526 Background: Archival tissues from patients with known clinical history have become an invaluable resource for gene expression analysis for tumor classification and clinical outcome prediction. These samples may be studied using qPCR, but RNA extracted from FFPE tissues is often significantly degraded, making them difficult to use in microarray analyses. Methods: We have developed the DASL assay, a flexible, sensitive and reproducible expression profiling method for parallel analysis of hundreds of genes in FFPE samples using universal bead arrays. The method can tolerate partial RNA degradation because it does not depend on an intact poly-A tail for cDNA synthesis. Tissue- and cancer-specific expression profiles have been obtained with as little as 50 ng total RNA derived from FFPE tissues that had been stored up to 11 years (Bibikova, et al., Am. J. Pathol. 165:1790–1807, 2004). Results: We performed a profiling study of 174 benign or cancerous prostate FFPE tissues retrieved from the VA San Diego Healthcare System. A total of 512 prostate cancer-related genes selected from the literature were monitored at 3 probes per gene. Despite RNA degradation, highly reproducible expression profiles were obtained for these samples (average r2 = 0.98). Expression data were used to identify tumor- and stroma-specific genes, which were then included in a sample classification scheme. Resulting gene panels correlated with clinical parameters such as tumor type, tumor grade, therapeutic response, and patient survival. In additional studies using laser capture microdissection of FFPE tissues, increased expression of tumor- and stroma-specific genes were measured in the correct cell types, confirming the results based on heterogeneous tissues. Conclusions: Putative prostate cancer biomarkers were identified using the DASL expression assay and RNAs derived from FFPE patient samples. In combination with the high-throughput capability of the bead array-based platform, the DASL assay may be used to monitor hundreds of genes in hundreds of samples at once. These data suggest that FFPE tissue archives, with known patient outcomes, can now be accessed for microarray expression analysis. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Illumina Illumina

Serial analysis of ribosomal sequence tags (SARST): a high-throughput method for profiling complex microbial communities.

Two decades of culture-independent studies have confirmed that microbial communities represent the most complex and concentrated pool of phylogenetic diversity on the planet. There remains a need for innovative molecular tools that can further our knowledge of microbial diversity and its functional implications. We present the method and application of serial analysis of ribosomal sequence tags (SARST) as a novel tool for elucidating complex microbial communities, such as those found in soils and sediments. Serial analysis of ribosomal sequence tags uses a series of enzymatic reactions to amplify and ligate ribosomal sequence tags (RSTs) from bacterial small subunit rRNA gene (SSU rDNA) V1-regions into concatemers that are cloned and sequenced. This approach offers a significant increase in throughput over traditional SSU rDNA clone libraries, as up to 20 RSTs are obtained from each sequencing reaction. To test SARST and measure the bias associated with this approach, RST libraries were prepared from a defined mixture of pure cultures and from duplicate arctic soil DNA samples. The actual RST distribution reflected the theoretical composition of the original defined mixture. Data from duplicate soil libraries (1345 and 1217 RSTs, with 525 and 505 unique RSTs, respectively) indicated that replication provides a strongly correlated RST profile (r(2) = 0.80) and division-level distribution of RSTs (r(2) = 0.99). Using sequence data from abundant soil RSTs, we designed specific primers that successfully amplified a larger portion of the SSU rDNA for further phylogenetic analysis. These results suggest that SARST is a powerful approach for reproducible high-throughput profiling of microbial diversity amenable to medical, industrial or environmental microbiology applications.

Suitability of free choice profiling for assessment of orange-based carbonated soft-drinks

A Free Choice Profiling test was carried out to find out whether assessors with a basic sensory knowledge are able to characterise flavour and odour of orange-based lemonades. The experiment was run with eight different samples of orange-based soft drinks in four replicates. Generalized Procrustes Analysis combined with Principal Component Analysis were used to analyse and plot the data. The analysis of the four separate replicates showed, that the assessors were not able to reproduce their description of the sensory characteristics suitably, there were relatively large differences between the assessments of the same products by the same assessors in the different replicates. Even for this data, however, the permutation test showed a highly significant group average configuration. This was mainly due to differences between the replicates. But a large residual variance remained. Only about 30% of the variability could be explained by the group average configuration, and only about 12% of the total variance could be explained by the first two dimensions of it. When the four assessments of each product were averaged to stabilize the data, 60% of the total variance could be explained by the group average configuration and the first two dimensions of it could explain 30% of the total variability. Therefore, at least a coarse interpretation of the consensus space could be made. Our general finding was that profiling of orange-based lemonades by nearly untrained assessors appears to be useful but should be considered with some reserve. It seems that the differences between the products were too subtle for a reproducible profiling by these methods. However, some interpretable results on the structure of the products and on the variables could be gained.

Proteomic evaluation of archival cytologic material using SELDI affinity mass spectrometry: potential for diagnostic applications.

Proteomic studies of cells via surface-enhanced laser desorption/ionization spectrometry (SELDI) analysis have enabled rapid, reproducible protein profiling directly from crude samples. We applied this technique to archival cytology material to determine whether distinct, reproducible protein fingerprints could be identifiedfor potential diagnostic purposes in blinded specimens. Rapid Romanowsky-stained cytocentrifuged specimens from fine-needle aspirates of metastatic malignant melanoma (with both known cutaneous primary and unknown primary sites), clear cell sarcoma, and renal cell carcinoma and reactive effusions were examined using the SELDI technology. A unique characteristic fingerprint was identified for each disease entity. Fifteen "blinded" unknown samples then were analyzed. When the protein profilefingerprints were plotted against the known fingerprints for the aforementioned diagnoses, the appropriate match or diagnosis was obtained in 13 (87%) of 15 cases. These preliminary findings suggest a substantial potential for SELDI applications to specific pathologic diagnoses.

Developmental toxicology of cadmium in living embryos of a stable transgenic zebrafish line.

The toxic effects of cadmium and other heavy metals have been well established, and many of these and other environmental pollutants are known to be embryotoxic or teratogenic. However, it has proven difficult to identify individual cells that respond to toxicants among the wide range of cell populations in an intact animal, particularly during early development when cells are continually changing their molecular and physiologic characteristics as they differentiate. Here we report the establishment of an in vivo system that uses hsp70 gene activation as a measure of cadmium toxicity in living early larvae of transgenic zebrafish carrying a stably integrated hsp70-enhanced green fluorescent protein (eGFP) reporter gene. We demonstrate that eGFP expression in this strain of fish acts as an accurate and reproducible indicator of cell-specific induction of hsp70 gene expression. Furthermore, the transgene responds in a dose-dependent manner at concentrations similar to those observed for morphologic indicators of early-life-stage toxicity and is sensitive enough to detect cadmium at doses below the median combined adverse effect concentration and the median lethal concentration. The stable nature of this transgenic line should allow for extremely rapid and reproducible toxicologic profiling of embryos and larvae throughout development.

Selective etching during the electrochemical C-V profiling of PM-HEMTs

In this study we investigate the appearance of etch pits and their effect on the ECV doping profiles of PM-HEMT structures grown by MOCVD and MBE. In some samples, etch-pits were observed after the etching of only 20 nm of the top layer. The observed pits had a circular pattern with diameter ranging from 2 to 25 μm. Study of these patterns by atomic force microscopy (AFM) showed that they are holes with a depth varying from 0.3 to 2 μm. The contribution of this hole area in the capacitance measurement during the ECV process results in the reduction and broadening of the observed peaks and in non reproducible profiling across the wafer. The dependence of the density and dimensions of the etch-pits on the etching depth are also presented.

Applications of fast protein liquid chromatography TM in the separation of plasma proteins in urine and cerebrospinal fluid.

Fast Protein Liquid Chromatography TM (FPLC), in which an anion-exchange column is used, provides rapid separation and reproducible profiling of the plasma proteins in urine and cerebrospinal fluid (CSF). Chromatographic separation of the proteins takes 1 h for urine specimens and 45 min for CSF. The elution sequence from the anion-exchange column is similar to the electrophoretic mobility. Individual proteins have the same retention times independently of which type of specimen is used. The elution characteristics of 21 plasma proteins have been identified. We illustrated some applications of this system, including the profiling of tubular protein-uria, the isolation of Bence Jones proteins from urine, and the investigation of hemoglobin-derived products in the CSF.

Applications of Electrochemical Methods for Semiconductor Characterization: I . Highly Reproducible Carrier Concentration Profiling of VPE “Hi‐Lo”

Factors which influence the accuracy and reproducibility of semiconductor carrier concentration profiling via capacitance‐voltage measurements and dissolution at an electrolytic Schottky barrier are critically examined. Attention is centered upon the capacitance contributions from the edge regions of the electrolyte contact area. It is shown that several properties of the electrolyte, and the measurement frequency, as well as the physical means of electrolyte confinement have an important influence. In particular, a suitable combination of electrolyte conductivity, wetting, and dissolution behavior can be met by 0.1M Tiron, for the profiling of using the “Post Office Profile Plotter.” Examples are given which demonstrate the high degree of reproducibility, both short‐ and long‐term, attainable for the profiling of “hi‐lo” structures. Optimization of the approach for other semiconductor materials, for which an “ideal” electrolyte may not be available, is also discussed.