2.1 Sample collection
The samples used for this study came from an institutional review board-approved project investigating organophosphate exposure. Blood samples were collected in lithium heparin tubes. RBCs were separated from plasma by centrifugation at 1285×g for 10 min at 4°C. Plasma samples were stored at −80°C until use. The RBCs were washed 3 times with PBS and then frozen at −80°C. The experiments reported here made use of blood fractions obtained from non-exposed control subjects. The samples were exposed in vitro to the chemically synthesized metabolic intermediates.
Plasma samples were incubated with 5 µM CBDP for 1h at room temperature. We performed a concentration dependence inhibition of BChE using plasma and different concentrations of CBDP, with a 10 min incubation (). For the MS characterization of adducts, we used 5 µM CBDP and a 1h incubation to insure complete inhibition of the BChE, with activity determined before and after incubation with CBDP.
Concentration dependence of a. CBDP inhibition of human plasma BChE, and b. PSP inhibition of human pure APH; following a 10 min incubation at ambient temperature. Results are represented as U/L or U/mg (U = µmol/min).
An analogue of CBDP, phenyl saligenin phosphate (PSP), was used to inhibit RBC APH, as described previously (Kim et al., 2010
). The concentration dependence of PSP inhibition of APH is shown in . For the MS characterization of adducts, 25 µM PSP was used with a 1h incubation to insure complete inhibition of the APH, with activity determined before and after the incubation.
CBDP (99.5% pure, CAS 1222-87-3) was custom synthesized by Starks Associates, Buffalo, NY, USA. PSP was a generous gift of Dr. Marion Ehrich.
2.2 BChE and APH purification
We have developed single-step immunomagnetic bead-based protocols (IMB) for purifying both plasma BChE and RBC APH to high levels of purity starting with just 200 µL of plasma or packed RBCs. For both protocols, we used magnetic beads (Invitrogen, Carlsbad, CA, USA), coupled to either mouse monoclonal anti-human plasma BChE antibodies (Thermo Fisher Scientific, Waltham, MA, USA) or in-house biotinylated, affinity purified polyclonal anti-human RBC APH antibodies (generated in rabbits by Cocalico Biologicals Inc., Reamstown, PA, USA—this company maintains a current USDA research license and a current Animal Welfare Assurance from the NIH’s Office of Laboratory Animal Welfare). We used 2.5% v/v (0.4 M) acetic acid to elute the antibody bound proteins from the IMBs.
We tested the efficiency of the IMB protein capture by activity assays of the samples before and after incubation with the beads. Enzyme activity was reduced by approximately 85–90%. All procedures were performed in Protein LoBind Tubes (Eppendorf, Hamburg, Germany).
The purified proteins were analyzed by gel electrophoresis using SDS-PAGE gels (NuPAGE® 4–12% BisTris Gel, Invitrogen, Carlsbad, CA, USA), stained with the Pierce® Silver Stain kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) or with the Imperial Protein Stain solution (Thermo Fisher Scientific Inc.).
2.3 Determination of BChE and APH activity
Plasma BChE activity was measured as described by Ellman (Ellman et al., 1961
), using 1 mM butyrylthiocholine in 0.1 M phosphate buffer pH 8.0 at 25°C. The reaction was started by the addition of 100 µL of diluted BChE to 100 µL of substrate solution and was monitored continuously at 405 nm over 3 min at 25°C.
Since APH has esterase activity, we adapted a protocol from Mastropaolo (Mastropaolo and Yourno, 1981
) to assay the RBC APH activity. A 0.73 M stock solution of alpha-naphthyl butyrate (ANB) in methanol was diluted to 0.2 mM with 20 mM Tris pH 7.0. The reaction was initiated by the addition of 200 µL of substrate solution to the sample, and was monitored continuously at 235 nm over 4 min at 25°C. We have also used the APH specific substrate N-acetyl-L-alanyl-p-nitroanilide (AcAlaPNA) to measure APH activity as described (Quistad et al., 2005
The assays were carried out in a SPECTRAmax® PLUS Microplate Spectrophotometer (Molecular Devices, Sunnyvale, CA). The AcAlaPNA was obtained from Bachem (Torrance, CA, USA). The other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).
2.4 Mass spectrometry protocols
Samples were analyzed by high resolution MS with the aim of detecting and quantifying active site serine modifications.
Samples obtained from IMB protocol were digested with 1 µg of chymotrypsin (Promega, Madison, WI, USA) for 2 h at 37°C and loaded onto an LC-MS/MS system nanoAcquity (Waters Corporation, Milford, MA, USA)/LTQ-FT (Thermo Fisher Scientific, Waltham, MA, USA). The system was equipped with a 30 cm column of 75 µm I.D. fused silica capillary pulled to a 5 µm I.D. tip using a Sutter Instruments P-2000 CO2 laser puller. The analytical column was packed with C-12 reversed phase chromatography material (Phenomenex, Jupiter 4µ, Proteo 90 Å) using an in-house constructed pressure bomb and compressed helium gas. Peptides were eluted using a 60 min water:acetonitrile gradient. SEQUEST was used for the databank search using the following dynamic modifications: S (Serine): 170.0138 (cresyl phosphoserine) and 79.9668 (phosphoserine) for BChE.