2-(2-cresyl)-4H-1-3-2-benzodioxaphosphorin-2-oxide (CBDP) was a generous gift from Wolf-Dietrich Dettbarn (Vanderbilt Univ.) and David E. Lenz (US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD). The CBDP (99.5% pure) was custom synthesized by Starks Associates (Buffalo, NY, USA). CBDP is also known as cresyl saligenin phosphate, cyclic tolyl saligenin phosphate, and saligenin cyclic-o-tolyl phosphate (SCOTP). The CAS number is 1222-87-3. CBDP was dissolved in acetonitrile to 100 mM and stored at −80 °C.
The following were from Sigma-Aldrich, St. Louis, MO: porcine pepsin (P-6887), S-butyrylthiocholine iodide (B-3253), 5,5′-dithiobis(2-nitrobenzoic acid) (D-8130), glycolic acid (Fluka 50590), formic acid (Fluka 94318), and 2, 5-dihydroxybenzoic acid (DHB) (Fluka 85707 and Acros 165200050).
The following were from Fisher Scientific, Fair Lawn, NJ: sequencing grade modified trypsin (Promega V5113), trifluoroacetic acid (A11650) and acetonitrile (BP1170-4).
Sources of the following reagents and their catalog numbers are as follows. Titansphere bulk media 5 micron (GL Sciences, Inc. 1400B500), Pro-Q Diamond phosphoprotein gel stain (Invitrogen, P33301), Amicon stirred cell 10 ml capacity (Millipore model 8010) with YM30 membrane (Millipore 13712), and Q-Sepharose fast flow (Amersham Pharmacia Biotech 17-0510-04).
Butyrylcholinesterase, for limit of detection studies and for phosphoprotein staining, was purified from outdated human plasma as described (Lockridge et al., 2005
). Double distilled water was from an in-house still. Procainamide Sepharose was synthesized with a 6-carbon spacer arm by Y. Ashani (Grunwald et al., 1997
Blood from jet airplane passengers
Subjects were healthy adult volunteers who donated blood within 48 h of disembarking from a jet airplane. There were no other criteria for selection of subjects. The 9 females and 3 males ranged in age from 25 to 68 years. All were college educated, non-smokers, not obese, who worked at white collar jobs unrelated to the airline industry. Many of our subjects traveled on a jet airplane once every three months. Subjects completed a questionnaire about possible toxic symptoms associated with travel on their flight. They also provided information on the destination and duration of travel, and donated 50 ml (4 tablespoons) of blood under a protocol approved by The Institutional Review Board of the University of Nebraska Medical Center. Blood was drawn into 10 red cap tubes (no anticoagulant) or into 10 green cap tubes (heparin anticoagulant) or into 10 lavender cap tubes (EDTA anticoagulant). A total of about 25 ml serum or plasma was recovered. Subjects donated blood within 24 to 48 hours of disembarking from a jet airplane. Control subjects had not traveled on a jet airplane for 3 to 15 months at the time they donated blood.
Assay of butyrylcholinesterase activity
Butyrylcholinesterase activity was measured with 1 mM butyrylthiocholine and 0.5 mM dithiobisnitrobenzoic acid in 0.1 M potassium phosphate buffer pH 7.0 at 25 °C. The change in absorbance at 412 nm was monitored in a Gilford spectrophotometer in 1 cm quartz cuvettes. The extinction coefficient for the product was 13,600 M−1cm−1. One unit of butyrylcholinesterase activity was defined as the amount that hydrolyzes 1 µmol of butyrylthiocholine in 1 min.
Determination of butyrylcholinesterase protein concentration and purity
Protein concentration was estimated from absorbance at 280 nm. To calculate the purity of butyrylcholinesterase we used an absorbance at 280 nm of 1.8 for a solution containing 1 mg/ml protein and a value of 720 units/mg as the specific activity of 100% pure butyrylcholinesterase (Lockridge et al., 2005
). Plasma butyrylcholinesterase is a tetramer of four identical subunits. The subunit molecular weight of human butyrylcholinesterase is 85,000 Da. The concentration of butyrylcholinesterase subunits in human plasma is about 4 mg per liter, or 50 nanomolar. The butyrylcholinesterase in plasma has to be purified about 12,000 fold to achieve a specific activity of 720 units/mg. The 12,000-fold purification requirement was calculated by using 3 units/ml as the average butyrylcholinesterase activity in plasma, and 50 mg/ml as the average protein concentration in plasma.
Purification of butyrylcholinesterase
Highly purified butyrylcholinesterase was prepared from 70 Liters of outdated human plasma as described (Lockridge et al., 2005
). The highly purified butyrylcholinesterase was used for the experiments in and . It was also tested for the presence of adducts on butyrylcholinesterase.
Figure 2 MALDI-TOF mass spectra of control unmodified butyrylcholinesterase and CBDP-butyrylcholinesterase digested with pepsin. A) Highly purified human butyrylcholinesterase with a specific activity of 518 units/mg and a concentration of 1.7 mg/ml was digested (more ...)
Figure 6 Polyacrylamide gel staining for phosphoproteins and total protein. Panel A, nondenaturing polyacrylamide gel stained for phosphoproteins with Pro-Q Diamond: Lanes A1 and A2; 10 and 5 µl of human plasma treated with CBDP to inhibit 100% of the (more ...)
Butyrylcholinesterase from individual jet airplane travelers was purified from about 25 ml of serum or plasma. The first step was dialysis against 2 × 4 Liters of 20 mM sodium acetate, 1 mM EDTA pH 4.0 (pH 4 buffer) at 4°C. Dialysis was performed in 22 mm diameter dialysis tubing with 12 000 MW cutoff (Spectrapor 3787-D32). During dialysis a heavy precipitate appeared which was removed by centrifugation. After dialysis the volume of serum or plasma typically increased by 2 ml. All chromatography steps were conducted at room temperature and were completed in one day. The clarified serum or plasma (approximately 27 ml) was loaded onto a 5 ml column of Q-Sepharose Fast Flow packed in a 15 ml (10 × 195 mm) Pharmacia column. The ion exchanger had been equilibrated by overnight washing with 1 Liter of pH 4 buffer. Yellow color and a large quantity of protein eluted during sample loading. The column was washed with 150 to 200 ml of pH 4 buffer until the absorbance at 280 nm of the eluant was approximately 0.03. A blue band of ceruloplasmin was visible at the top of the column at the start of the buffer wash, but it eluted during washing with buffer. Butyrylcholinesterase eluted with a linear 100 ml gradient of zero to 0.2 M NaCl in pH 4 buffer. Three ml fractions were collected. The butyrylcholinesterase began to elute when the NaCl concentration was 0.05 M (Lockridge et al., 2005
). The purest butyrylcholinesterase eluted early, in fractions 7–10, occupying a volume of 9 to 12 ml. Later fractions contained 10-fold less pure butyrylcholinesterase.
The second step of the purification was on a 2 ml procainamide Sepharose affinity column packed in a 7 ml (10 × 95 mm) Pharmacia column, equilibrated with 20 mM TrisCl, 1 mM EDTA pH 7.5 at room temperature. TrisCl was used rather than phosphate buffer because phosphate ions compete with phosphorylated peptides for binding to titanium oxide beads in the enrichment step that precedes mass spectrometry. Fractions from the ion exchange column containing the purest butyrylcholinesterase were pooled and the pH adjusted from 4 to 7 by adding 0.19 ml of 1 M NH4HCO3 per 3 ml butyrylcholinesterase. The sample was loaded onto the procainamide affinity gel. The affinity gel was washed with 20–30 ml of 20 mM TrisCl, 1 mM EDTA pH 7.5 and 20 ml of 0.2 M NaCl in pH 7.5 buffer. Butyrylcholinesterase was eluted with 1 M NaCl in pH 7.5 buffer. The purest BChE (15 to 20% pure) eluted in the first 4 ml, the next 2 ml typically contained 9% pure BChE, and the last 4 ml contained 3% pure BChE.
The purity of butyrylcholinesterase following ion exchange at pH 4 and procainamide affinity chromatography varied for each sample. With most samples we achieved an 1800-fold increase in specific activity to 15% purity, but the range varied from 2 to 30% purity. The highest purity was achieved when the serum or plasma volume was at least 30 ml and when the starting butyrylcholinesterase activity was 3 units per ml. A low 2% purity was achieved when the plasma volume was 17 ml and the starting plasma butyrylcholinesterase activity was 1.3 units per ml. Generally, about 30% of the starting activity could be recovered at 15% purity. In some cases the yield was 50%. Similar purification yields were obtained for serum, heparin plasma, and EDTA plasma. Percent purity was calculated from absorbance at 280 nm and activity per ml using the information that 100% pure BChE has a specific activity of 720 units/mg and a 1 mg/ml solution of BChE has an absorbance at 280 nm of 1.8 (Lockridge et al., 2005
). For example, a 15% pure solution of BChE with an activity of 60 units/ml has an absorbance of 1.0 at 280 nm.
Butyrylcholinesterase dialysis and concentration
The two-step purified butyrylcholinesterase was concentrated in a 10 ml Amicon stirred cell with a YM30 membrane. The use of the YM30 membrane in the Amicon stirred cell resulted in no loss of butyrylcholinesterase activity, whereas other concentrating devices such as the Amicon centrifugal filter 10,000 MWCO (UFC901024) resulted in significant losses of activity. The buffer was changed to 10 mM NH4HCO3, 0.01 % (w/v) sodium azide, pH 8.1 by diluting and re-concentrating 3 times. Changing the buffer and removing the salt was necessary to promote ionization of the peptides in the mass spectrometer. The final volume of the concentrated butyrylcholinesterase was 0.1 ml and the activity was about 120 units/ml. The fact that we found phosphorylated butyrylcholinesterase peptide in our partially purified butyrylcholinesterase samples means that phosphorylated butyrylcholinesterase copurified with unmodified butyrylcholinesterase in our protocol. Thus, we do not expect the ratio of phosphorylated to unmodified butyrylcholinesterase to change after purification.
When only 0.05% of the butyrylcholinesterase was phosphorylated, about 12 units of partially purified butyrylcholinesterase (16.6 µg) were needed for mass spectrometry detection of phosphorylated butyrylcholinesterase. The final degree of purity was not critical, though it was important to remove albumin, the blood coagulation proteins, and proteins that precipitate at pH 2. The pH is lowered to 2 for pepsin digestion. Our protocol would work for smaller volumes of blood if the level of exposure were high. For example, we have detected other organophosphorus and carbamate adducts on butyrylcholinesterase using 1 or 2 ml plasma from suicide and murder victims (Li et al., 2009
; Li et al., 2010
). In those cases 60–80% of the butyrylcholinesterase was inhibited.
Stability of phosphorylated butyrylcholinesterase
Two of the partially purified butyrylcholinesterase samples from jet airplane passengers were stored at 4°C in 10 mM ammonium bicarbonate, 0.01% sodium azide pH 8.3 for one year before they were digested with pepsin. Both samples were found to be positive for the phosphorylated butyrylcholinesterase peptide. This demonstrates that the phosphorylated butyrylcholinesterase adduct is stable and that it does not spontaneously dephosphorylate. Phosphorylated butyrylcholinesterase was found in butyrylcholinesterase purified from serum as well as plasma, suggesting that phosphatases in blood do not dephosphorylate the butyrylcholinesterase adduct.
Pepsin digestion of butyrylcholinesterase
The pH of partially purified butyrylcholinesterase in 0.1 ml of 10 mM NH4HCO3, 0.01 % sodium azide was adjusted to pH 2 by adding 1 µl of 25% trifluoroacetic acid. A fresh solution of 5 mg/ml pepsin in 5% formic acid was prepared just before use. Pepsin dissolved with difficulty to make an opalescent solution. The butyrylcholinesterase was digested with 5 µl pepsin (25 µg) at 37 °C for 2 h. Digestion was stopped by inactivating the pepsin either by 5 min of incubation in a boiling water bath, or by adjusting the pH of the digest to 7.
The pepsin digestion step was the most difficult to optimize. When attempts were made to simplify the purification by omitting the pH 4 ion exchange step and purifying only on procainamide affinity gel, acidification prior to addition of pepsin caused the butyrylcholinesterase solution to solidify into an indigestible clot. A second problem was that pepsin partially precipitated out of solution at 1 mg/ml in 10 mM hydrochloric acid when stored frozen in 10 µl aliquots (a common method for preparing pepsin). The precipitate was not visible to the eye, but the effect on digestion was dramatic in that the target peptide was not generated. A third problem was pH. As noted above, the purity of butyrylcholinesterase preparations ranged from 2% to 30% depending on the volume and activity of the starting plasma and on the extent to which side-fractions were pooled with the main fractions. Less pure preparations required more acid to lower the pH to 2 and required more pepsin for proper digestion. The amount of pepsin to add was calculated as follows. Twelve units of butyrylcholinesterase = 16.6 µg butyrylcholinesterase protein. A 17% pure preparation therefore contains a total of 98 µg protein (16.6 µg/0.17 = 98 µg). The desired ratio of total protein to pepsin is 4 to 1 on a weight basis (98 µg/4 = 25). Therefore, 25 µg of pepsin were required to digest 12 units of 17% pure butyrylcholinesterase.
Enrichment of phosphorylated peptide by binding to titanium oxide
Phosphorylated butyrylcholinesterase peptide was enriched and concentrated by the method of Jensen and Larsen (Jensen and Larsen, 2007
beads from GL Sciences Inc. (Torrance, CA, USA) were manually packed into empty pipette tips (that previously contained TopTip POROS R-2 1–10 µl part no TTIPR2.96 from the Glygen Corp., Columbus, MD, USA) to a bed height of 3 mm (2.2 mg TiO2
). The Glygen tips come with an adapter that holds the packed pipette tip in a standard 1.5 ml microfuge tube, allowing solvent to be centrifuged through the tip. Sample and solvents were centrifuged through the TiO2
microcolumn at a speed of 4000 rpm in a Sorvall MC12V microfuge (1500 × g). Higher speeds extruded the beads out of the tip. The TiO2
microcolumn was conditioned with 75 µl of 75% acetonitrile, 1% trifluoroacetic acid and washed with 75 µl loading buffer (1 M glycolic acid in 80% acetonitrile, 5% trifluoroacetic acid). Before the sample was loaded onto the microcolumn, the pepsin-digested butyrylcholinesterase (100 µl) was diluted with an equal volume of loading buffer and centrifuged at 12,000 rpm to remove turbid material which could clog the beads. The clarified sample was centrifuged through the microcolumn at 4000 rpm. The microcolumn was washed with 75 µl loading buffer, followed by 3 × 75 µl of 75% acetonitrile, 1% trifluoroacetic acid. Phosphorylated peptides were eluted with 2 × 20 µl of 0.4 M ammonium hydroxide, 30% acetonitrile. Fractions were combined and the volume was reduced to about 1 µl in a SpeedVac. The sample was mixed with 2 µl of 2,5-dihydroxybenzoic acid matrix (20 mg/ml DHB in 50% acetonitrile, 0.1% trifluoroacetic acid, 1% phosphoric acid) and spotted on a Maldi plate. If no signal was obtained, the sample was dried completely, dissolved in 5 µl of 50% acetonitrile, 1% trifluoroacetic acid, and spotted on a MALDI plate. After the spot was dry, it was overlaid with DHB matrix.
MALDI mass spectra were acquired on a MALDI–TOF/TOF 4800 mass spectrometer (Applied Biosystems, Framingham, MA, USA). Data collection was controlled by 4000 Series Explorer software (version 3.5). Mass spectra were taken in negative reflector mode using delayed extraction (625 nsec) and default calibration. The mass spectrometer was calibrated in positive mode against bradykinin (904.47 m/z), angiotensin 1 (1296.68 m/z), Glu-fibrinopeptide B (1570.68 m/z), adrenocorticotropic hormone (ACTH) 1–17 clip (2093.09 m/z), ACTH 18–39 clip (2465.20 m/z), and ACTH 7–38 clip (3657.96 m/z) (Cal Mix 5 from Applied Biosystems). Each spectrum was the average of 500 laser shots taken with the laser energy adjusted to 5000 volts. MS/MS fragmentation spectra were taken using postsource decay in positive mode at 1 kilovolt collision energy in the absence of collision gas and with metastable ion suppression on. Each spectrum consisted of 500 laser pulses taken with the laser energy adjusted to yield optimal signal-to-noise. MS/MS calibration used the fragmentation spectrum of angiotensin 1. Spectra were analyzed with Data Explorer Software.
Limit of detection of phosphorylated butyrylcholinesterase
Partially purified butyrylcholinesterase with a specific activity of 173 units/mg (24% pure) was used for the experiment to determine the limit of detection of phosphorylated butyrylcholinesterase. This purification level was selected because it was similar to the purity of butyrylcholinesterase processed from plasma of jet airplane passengers. An aliquot of this butyrylcholinesterase, in 10 mM NH4HCO3, 0.01% sodium azide with an activity of 120 units/ml, was treated with CBDP to inhibit 100% of the butyrylcholinesterase activity. The CBDP-butyrylcholinesterase was digested with pepsin. CBDP does not inhibit pepsin, so there was no need to remove excess CBDP. Control unmodified butyrylcholinesterase from the same lot was also digested with pepsin. The digested CBDP-butyrylcholinesterase was added to the digested control butyrylcholinesterase so that the proportion of CBDP-butyrylcholinesterase ranged from 0.05 to 5%. The total amount of butyrylcholinesterase in each mixture was 16.6 µg in a volume of 0.1 ml. The digests were enriched for phosphorylated peptides by binding to TiO2 and eluted with 0.4 M NH4OH, 30% acetonitrile. The eluted peptides were analyzed in the MALDI-TOF mass spectrometer. The phosphorylated, active-site, peptic peptide of butyrylcholinesterase (FGEpSAGAAS) has a mass of 874.3 Da in negative mode.
Phosphorylated proteins stained with Pro-Q Diamond
Polyacrylamide 4–30% gradient gels 0.75 mm thick were prepared in a Hoefer SE600 gel apparatus and run at a constant voltage for 3000 volt-hours (150 V for 20 h) at 4°C. Phosphorylated proteins were visualized by staining with Pro-Q Diamond followed by measurement of fluorescence on a Typhoon 9410 Imager (GE Healthcare Life Sciences) with 532 nm excitation and 580 nm longpass emission filters. Gels were counterstained with Coomassie Blue R-250.