Quantitative Proteomic Analysis of Human Aqueous Humor Using iTRAQ 4plex Labeling

Abstract

iTRAQ 4plex method enables multiplexing of up to four samples for simultaneous quantitation to improve sensitivity and scope of proteomic analysis. Here, we describe iTRAQ 4plex labeling of human aqueous humor specimens followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and data analysis for peptide identification and quantitation using Proteome Discoverer software. The protocol can be applied for other animals as well; however, pooling of specimens may be required to obtain sufficient amount of protein for labeling.

Liquid Biopsy Proteomics in Ophthalmology

“…However, the small volume of AH or vitreous samples represents a critical challenge for mass spectrometry-based studies. One solution may be to pool samples, but this then requires extensive labeling techniques to distinguish individual samples . Another challenge is that the high levels of albumin and immunoglobulins can mask lower abundant proteins, a problem that may be addressed by technologies that combine nanoparticle protein coronas with liquid chromatography-mass spectrometry .…”

“…One solution may be to pool samples, but this then requires extensive labeling techniques to distinguish individual samples. 43 Another challenge is that the high levels of albumin and immunoglobulins can mask lower abundant proteins, a problem that may be addressed by technologies that combine nanoparticle protein coronas with liquid chromatography-mass spectrometry. 44 An aptamer-based assay recently demonstrated its potential for high-resolution proteomics in AH and vitreous specimens.…”

Quantitative proteomic analysis after neuroprotective MyD88 inhibition in the retinal degeneration 10 mouse

“…ITRAQ Reagents (8‐plex) (Sciex #4390733) were added to samples and incubated at room temperature for 2 h, following our previous study. 21 The reaction was quenched by adding 100 µl of LC‐MS grade water and incubated for another 30 min. All samples were combined into one tube, dried in a speed vacuum concentrator, washed three times with 100 µl of LC‐MS grade water and then stored dry at 20°C until analysis by high‐performance liquid chromatography tandem mass spectrometry (LC‐MS/MS).…”

“…ITRAQ Reagents (8-plex) (Sciex #4390733) were added to samples and incubated at room temperature for 2 h, following our previous study. 21 The reaction was quenched by adding 100 µl of LC-MS grade water and incubated for another 30 min.…”

The effect of extrinsic Wnt/β‐catenin signaling in Muller glia on retinal ganglion cell neurite growth

“…Proteins were analysed in the conditioned media following our previously published papers (Edwards, Mesa, Vazquez-Padron, Kowalski, & Bhattacharya, 2017;Trzeciecka, Pattabiraman, Piqueras, Toris, & Bhattacharya, 2018) with the following modifications. Proteins were precipitated from conditioned media samples by adding 4 volumes of acetone and incubating overnight at -20°C.…”