An Internal Calibration Method for Protein-Array Studies

Abstract

Nuisance factors in a protein-array study add obfuscating variation to spot intensity measurements, diminishing the accuracy and precision of protein concentration predictions. The effects of nuisance factors may be reduced by design of experiments, and by estimating and then subtracting nuisance effects. Estimated nuisance effects also inform about the quality of the study and suggest refinements for future studies.We demonstrate a method to reduce nuisance effects by incorporating a non-interfering internal calibration in the study design and its complemental analysis of variance. We illustrate this method by applying a chip-level internal calibration in a biomarker discovery study. The variability of sample intensity estimates was reduced 16% to 92% with a median of 58%; confidence interval widths were reduced 8% to 70% with a median of 35%. Calibration diagnostics revealed processing nuisance trends potentially related to spot print order and chip location on a slide. The accuracy and precision of a protein-array study may be increased by incorporating a non-interfering internal calibration. Internal calibration modeling diagnostics improve confidence in study results and suggest process steps that may need refinement. Though developed for our protein-array studies, this internal calibration method is applicable to other targeted array-based studies.

Oxidatively modified proteins as plasma biomarkers in breast cancer

“…Immediately before analysis, the samples were thawed on ice and centrifuged at 15,000 g for 20 min at 4 °C to remove trace precipitates. The supernatant was diluted 5-fold in 0.1% BSA/PBS containing 1000 pg/mL of green fluorescent protein, which was used as the antigen in a calibrant sandwich ELISA to identify and normalize any chip-to-chip bias [10, 11]. The individual capture antibodies (listed in Supplementary Table 1) are typically saturated by low ng/mL concentrations of antigen, and we routinely dilute plasma samples 500-fold in order to get antigen concentrations in the usable range of the standard curve for the unmodified proteins.…”

“…After processing, the microarray slides were imaged with a fluorescent laser scanner (ScanArray Express HT, Perkin-Elmer, Downer Grove, IL, USA) and ScanArray Express software was used to quantify the fluorescent intensity of the spots. Data calibration across chips was undertaken using the data from the green fluorescent protein ELISA analysis using ProMAT Calibrator [10, 11] and then was processed using ProMAT [16]. We developed both of these programs as open-source programs in R that are freely available on the web (www.pnl.gov/statistics/ProMAT/).…”

High‐Throughput, Multiplexed Analysis of 3‐Nitrotyrosine in Individual Proteins

“…Capture antibodies, along with orientation spot A546 (0.1 mg/ml, Alexa Fluor 546 labeled goat anti-rabbit IgG), PBS (buffer control or blank), non-immune rabbit IgG (negative control for non-specific protein binding), and GFP antibody (used for a calibrant assay) (Daly et al, 2010) are printed on polylysine-coated glass slides that are preprinted with hydrophobic barriers to create 16 separate, but identical, wells on each slide (Gonzalez et al, 2008b).…”

“…Dilute the plasma samples 5-fold with 0.1% BSA/PBS. Spike a final concentration of 100 pg/ml GFP into each sample for evaluation of data quality and, if needed, normalization (Daly et al, 2010).…”

A semi-nonparametric mixture model for selecting functionally consistent proteins

“…Toward this end, microarray technology when adapted to proteins, are known as protein microarrays, and have been developed and widely used to assess the abundance of proteins [6-12]. The similarities between microarray technology as applied to gene expression [13], and as applied to protein abundance, are the same in that improved accuracy and precision, as well as design issues and normalization techniques for protein microarrays have been established [14,15]. …”