High dynamic range proteome imaging with the structured illumination gel imager

Abstract

A current challenge for proteomics is detecting proteins over the large concentration ranges found in complex biological samples such as whole-cell extracts. Currently, no unbiased, whole-proteome analysis scheme is capable of detecting the full range of cellular proteins. This is due in part to the limited dynamic range of the detectors used to sense proteins or peptides. We present a new technology, structured illumination (SI) gel imager, which detects fluorescently labeled proteins in electrophoretic gels over a 1 000 000-fold concentration range. SI uses computer-generated masks to attenuate the illumination of highly abundant proteins, allowing for long exposures of low-abundance proteins, thus avoiding detector saturation. A series of progressively masked gel images are assembled into a single, very high dynamic range image. We demonstrate that the SI imager can detect proteins over a concentration range of approximately 1 000 000-fold, making it a useful tool for comprehensive, unbiased proteome-wide surveys.

Mutant huntingtin disrupts mitochondrial proteostasis by interacting with TIM23

“…To assess the downstream impact of the mHTT-TIM23 association, which results in the previously demonstrated mitochondrial protein import inhibition (19), we performed 2D-DIGE comparing mitochondrial protein lysates from Q7 and Q111 cells labeled with fluorophores Cy3 and Cy5, respectively. After electrophoresis, SDS polyacrylamide gels were imaged to detect Cy3 and Cy5 signals separately (36). A representative 2D-DIGE image of Q7 and Q111 mitochondrial lysates is shown in Fig.…”

“…Green indicates Cy3-labeled Q7 sample; red indicates Cy5-labeled Q111 sample. n = 6 with reciprocally dye-labeled technical replicates for each n. The full 2D-DIGE gel image shown is a composite of 16 field-of-view camera shots(36). (B) Enlarged version of the cropped rectangle from A. Reproducible difference proteins are highlighted in white ovals.…”

Proteomic analysis reveals APC-dependent post translational modifications and identifies a novel regulator of β-catenin

“…1B) (Minden, 2012). This technique combined with a high dynamic range fluorescence imaging system can detect as little as 0.2 fmol of protein over a 100,000-fold concentration range (Minden, 2012;Van et al, 2014a). Difference-proteins can then be identified using liquid chromatography coupled to tandem mass spectrometry (LC-MS/ MS).…”

Reversible Click Chemistry Tag for Universal Proteome Sample Preparation for Top-Down and Bottom-Up Analysis

“…Binding of labeled proteins to TCO-beads was assessed by running the effluents and a load control on a 4−20% SDS-PAGE gel (BioRad) for 1 h at 120 V. Fluorescence images were acquired using a custom-built imager. 38 The amount of unbound protein remaining in the effluent was quantified according to the pixel intensity of the fluorescence images using ImageJ with the protein amount being calculated as a percentage of the load. 38…”

“…38 The amount of unbound protein remaining in the effluent was quantified according to the pixel intensity of the fluorescence images using ImageJ with the protein amount being calculated as a percentage of the load. 38…”