Highly sensitive detection of S-nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence

Abstract

S-nitrosylated proteins are biomarkers of oxidative damage in aging and Alzheimer's disease (AD). Here, we report a new method for detecting and quantifying nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF). Dylight 488 maleimide was used to specifically label thiol group (SH) after switching the S-nitrosothiol (S-NO) to SH in cysteine using the "fluorescence switch" assay. In vitro nitrosylation model-BSA subjected to S-nitrosoglutathione (GSNO) optimized the labeling reactions and characterized the response of the LIF detector. The method proves to be highly sensitive, detecting 1.3 picomolar (pM) concentration of nitrosothiols in nanograms of proteins, which is the lowest limit of detection of nitrosothiols reported to date. We further demonstrated the direct application of this method in monitoring protein nitrosylation damage in MQ mediated human colon adenocarcinoma cells. The nitrosothiol amounts in MQ treated and untreated cells are 14.8±0.2 and 10.4±0.5 pmol/mg of proteins, respectively. We also depicted nitrosylated protein electrophoretic profiles of brain cerebrum of 5-month-old AD transgenic (Tg) mice model. In Tg mice brain, 15.5±0.4 pmol of nitrosothiols/mg of proteins was quantified while wild type contained 11.7±0.3 pmol/mg proteins. The methodology is validated to quantify low levels of S-nitrosylated protein in complex protein mixtures from both physiological and pathological conditions.

Figure 1

(A) Schematic of fluorescence switch assay coupled with CGE-LIF for detecting and quantifying S-nitrosylated proteins. Fluorescence switch assay (Steps 1–3) includes blocking thiol group by MMTS, reducing nitrosothiol group with ascorbate and labeling reduced free thiol by Dylight 488 maleimide. Finally, labeled proteins are separated and analyzed by CGE-LIF.

Figure 2

CGE-LIF separation of nitrosylated BSA labeled with Dylight 488 maleimide. Separation, -570V/cm; hydrodynamic injection, 11kpa, 4s; sieving matrix, 20mM Tris, 20mM Tricine, 0.5% SDS, 15% dextran (65.5kDa), pH 8. Top trace and middle trace are offset on the y-axis for clarity. * — Dylight 488 maleimide, ^ — nitrosylated BSA.

Figure 3

Electropherograms of a series of dilution (1-, 2-, 4-, 10-fold) of nitrosylated BSA labeled with Dylight 488 maleimide. Other conditions are the same as in Fig.2. *—Dylight 488 maleimide, ^—nitrosylated BSA.

Figure 4

Electropherograms of extracted proteins from HT29 cells after fluorescence switch assay. Top and bottom traces represent MQ treated and untreated, respectively. All the experimental conditions are the same as in Fig 2. Top trace is offset in the y-axis for the clarity. *—Dylight 488 maleimide; migration window between a and b indicated nitrosylated proteins.

Figure 5

Electropherograms of cerebrum from 5-month-old transgenic (upper trace) and wild type (bottom trace) mice brain tissue. All the experimental conditions are the same as in Fig 2. Top trace is offset in the y-axis for the clarity. *—Dylight 488 maleimide; migration window between a and b indicated nitrosylated proteins.