2.4 PROTEIN MANIPULATIONS
2.4.7 DISULFIDE MAPPING PROTOCOL
2.4.7.1 DISULFIDE MAPPING OF EAS ISOFORMS.
the Structure and Ratios Ionized
The disu lfide mapping protocol requires that peptides be created from cyanylated protein species by digesting them ammonia and TCE P (Wu and Watson, 1 997). it is
necessary to predict both the structure and mass of each peptide that might be created. I used MOL® ISIS Draw (version 2 . 5) software to draw the structure of each peptide using the amino acid sequence of EAS as a g u ide (Mackay et al. , 200 1 ) . The software was then used to calculate the molecular m ass of each peptide (eq uivalent to the m/z value for the molecular ion).
Partial Reduction and
The partial reduction and cyanylation reaction was optimised em pirically as descri bed in section 5.2. 1 (see page 1 38) and the reaction conditions that gave the best yields of singly-reduced and cyanylated EAS isomers are reported in Table 1 5 (see page 1 40). However, the same general procedure (described below) was followed when I performed each partial-reduction and cyanylation reaction; the only parameters I altered were the reaction tem perature and the concentration of the reagents.
I n order to partially reduce and then cyanylate EAS , between 0 . 5 - 1 mg (0 . 07 �mal O . 1 3 �mol) of a RP-H PLC purified EAS isoform in 50 - 1 00 IJ L of a weakly acidic, denaturing-buffer consisting of 6 M guanidine hydrochloride dissolved in 0. 1 M citrate buffer, pH 3 . 0 . The citrate buffer solution was made by mixing 82 m L of an aqueous solution 0 . 1 M citric acid with 1 8. 0 mL of an aqueous solution of 0. 1 M trisodium citrate, fi ne pH adj ustments were m ade with 1 M solutions of either com ponent.
The partial reduction reaction was performed by add ing between 0 . 5 - 5 IJ L (depending o n the experiment) of an 1 M aqueous solution of tris(2-carboxyethyl) phosphine (TCEP, obtai ned from Pierce®) to the EAS solution and allowing it to react for 1 5 m i n at the desired tem perature (at either 1 1 °C or 25 °C). I m mediately afterward , between 20 IJL and 40 IJL of a cyanylation solution (1 M 1 -cyano-4- dimethylam i n o-pyridinium tetrafluoroborate (CDAP) d issolved in 0. 1 M citrate buffer) was added to the partly reduced EAS solution in order to cyanylate the protein's nascent sulfhydryls. After C DAP was added, the reaction solution was incubated at 25 °C for 30 m i n before the reduced and cyanylated EAS species were resolved by RP-H PLC .
Solutions of TCEP and C DAP were freshly prepared j ust prior to use, and vials containing these chem icals were kept tightly sealed and i n the presence of a desiccant at -20 °C until req uired . These precautions were necessary because although TCE P is more stable than OTT (Han and Han, 1 994; Getz et al. , 1 999), it is known that TCEP oxidises rapidly in the presence of certain buffering and chelati ng agents (Getz et al. , 1 999; Pierce®, 2003). When required, the extent of oxidation i n
Methods and Materials.
com mercial preparations of TCEP was monitored by spectroscopically measuring its ability to reduce 0.2 m mols of 5,5'-dithiobis(2-nitrobenzoic acid) (DTN B or Ellman's reagent) i n a procedure described by Han and Han ( 1 994). During these experi ments, equivalent amounts of OTT were used as a control.
RP-HPLC EAS Isomers.
The RP- H P LC protocol used to fractionate the partially reduced and cyanylated EAS isomers was optimised empirically as described in section 5.2. 1 . 3 (see page 1 40).
( 1 ) During the i n itial purification experiments, the reaction solution was de-salted by first diluting with Mi lli-Q® water as requ i red, applying the solution to an Amersham® pre-packed disposable PD-1 0 de-sa lting col u m n (a gravity flow colum n packed with Sephadex™ G-25 Medium), and then eluting the protein from the column with M i l l i-Q® water as instructed by the m a nufacturer. The eluate was freeze-d ried and then dissolved in 30 %(v/v) acetonitri le and 0 . 1 % (v/v) TFA before being applied to the Jupiter™ 250 mm x 1 0 mm column protected with the Jupiter™ 50 m m x 1 0 m m guard col u m n (see Table 4 for details). The reduced and cyanylated protein species were eluted using the g radient profi le described by Figure 1 4, except that the flow rate and upper pressure l i m it was adj usted to 0 . 5 mllmin and 1 20 bar respectively.
(2) The parameters of the purification protocol that I used to resolve the singly red uced and cyanylated EAS isomers that revealed the pattern of disulfide bonds in EAS were as follows. Reaction solutions prepared according to the "Final optim ised protocol" descri bed in Table 1 5 (page 1 40), were applied directly to a Amersham® � RPC C2/C 1 8 ST 4.6/1 00 R P-H PLC column protected with the Ju piter™ 50 m m x 1 0 m m g uard col u m n (see Table 4 for details), and resolved using the RP-HPLC gradient profile described by Figure 1 5. Each protein fraction was collected m anually as it el uted from the column.
The am monia-catalysed S-cyanylated peptide-cleavage phase of the disulfide mapping protocol methodology was carried out by adding 8 �L of either an ammonium hyd roxide ( 1 M NH40 H , pH 1 2) or denaturing a m monium hydroxide solution (6 M guanidine hydrochloride dissolved in 1 M NH40 H , pH 1 2) and allowed to react with EAS at 25 °C for 1 hr (Wu and Watson, 1 997) . I n order to achieve complete peptide cleavage i n 1 hr, both ammonium hydroxide sol utions m ust be at pH 1 2 : if this wasn't the case then the pH was adj usted with additional N H40H (Wu and Watson , 1 997).
FIGURE 14: RP-HPLC MOBILE PHASE GRADIENT: USED TO FRACTIONATE REDUCED AND CYANYLATED EAS ISOMERS WITH THE JUPITER 250 mm x 4.6 mm COLUMN.
CHEMST A TION PROGRAM DETAILS GRADIENT PROFILE2
Time of each Solvent RP-I-PLC Gradient PfC'!;Um Robpep..m Gradient Cohsm ftush
Programmed components in the 100
Change. mobile phase 1 - - - -·�
· · • · · Acetonitrile!TF A % A % C % 0
i
-•-waterfTFA o m in 0 95 5 :::(! 0 c 0 5 min 0 95 5 � c 45 min 0 30 40 � c 0 0 c •. so min 0 0 100 Q) > 20 0 55 m in 0 95 5 Cl) -· . . • 0Flow Rate Max Pressure Limit 10 15 20 25 40 45 55
os mLjmin 300 bar Retention Time (min)
1 Solvent A = 1 00% HPLC-9rade methanol. Solvent C = 0.1 %(v/v) TFA in Mer sterilised Milli-Q water. Solvent 0 = 0.1 %(v/v) in HPLC grade acetonitrile. '%A,' '%C,' and '%0' refer to the concentration of each component as a percentage of the total mobile phase. 2 The gradient profile depicts the RP-HPLC mobile phase gradient resulting from the ChemStation program. This program was used exclusively with the JupiterTM 250 mm x 1 0 mm column.This program was used exclusively with the JupiterTM 250 mm x 4.6 mm column.
FIGURE 15: RP-HPLC MOBILE PHASE GRADIENT USED TO FRACTIONATE REDUCED AND CYANYLATED EAS ISOMERS WITH THE p.RPC ST 4.6/100 COLUMN.
CHEMSTATION PROGRAM DETAILS GRADIENT PROFILE2
Time of each Solvent Gradlert Coll.nYl ftush
Programmed components in the 100 RP-I-IPLC Gra::iert Program RD.'V_HS.m
Change. mobile phase 1 •
• Acetonirtile/TF A % A % C % 0
�
-· -•-\11.8terfTFA ·-· 70 o min 5 75 20 c 0 "" s min 5 75 20 � 50 c Q) 70 min 5 0 95 u c 0 40 0 75 min 5 75 20 c Q) > 20 • 0 70 min 5 75 20 Cl) 10 0Flow Rate Max Pressu re Limit 0 10 20 30 40 50 60 70 80
osmL/min 30o bar Retention Time (min)
1 Solvent A = 1 00% HPLC-9rade methanol. Solvent C = 0. 1 %(v/v) TFA in filter sterilised Milli-Q water. Solvent 0 = 0. 1 %(v/v) in HPLC grade acetonitrile. '%A,' '%C,' and '%0' refer to the concentration of each component as a percentage of the total mobile phase. 2 The gradient profile depicts the RP-HPLC mobile phase gradient resulting from the ChemStation program. This program was used exclusively with the �RPC ST 4.6/100 column described in Table 4.
The ammonia was removed under vacuu m and either 2 IJL of 0 . 1 M aqueous TCEP (when the denaturing ammonium hydroxide solution was used) or 2 IJ L of 0 . 1 M OTT i n 0. 1 M Tris HCI buffer (pH 8.0) was mixed with the EAS reaction solution and allowed to reacted at 37 °C for 30 min. Adding these reducing agents had the effect
Methods and Materials.
of removing the residual disulfide bonds that linked the peptides generated during the preceding reactions.
Mass
The combination of peptide-cleavage and full-red uction reactions produces a
mixture of peptide fragments whose com position is dependent on the positions of the cyanylated cysteines in the partly reduced parent-protein. Two m ass spectrometry methods, ES I/MS, and Matrix-Assisted Laser Desorption/l onisation time-of-fl ight mass spectrometry (MALD I-TOF/MS) were used to analyse these peptide m ixtures.
ES I/MS analysis was carried out by Dwayne Jenson (Biological Chemistry, Hort Research) using a Finnigan LCQ™ Deca ion-trap mass spectrometer with a
Surveyor® liquid chromatography (LC) system, controlled using the Xcalibur®
(version 1 . 3) software that was also used for signal analysis and de-convolution. Samples analysed by ES I /MS were first dil uted in 1 : 1 mixture of acetonitrile and water with 0 . 1 %(v/v) acetic acid (added as an ion pairing agent) and applied to a Ju piter™ 5 1-J m C4 H PLC column '(the same model described i n Table 4) that had been attached to the Surveyor® LC System . The peptides were then resolved by RP H PLC. The Finnigan LCQ ™ Deca ESI/MS, mounted inline with the LC syste m , was able to analyse the sam ple components as they eluted from the column.
MALDI-TOF/MS ana lysis was performed with a M icromass Systems M@ldi™ LR mass spectrometer equipped with a 337 nm nitrogen UV Laser and was controlled using Mass lynx™ softwa re (version 3.5 for Microsoft Windows® NT). This device was based at the I nstitute of Fundamental Science s , Massey University, Palmerston North , New Zealand . M @ldi ™ LR mass spectrometers are equi pped with both linear-mode and reflectron-mode detectors and the time-to-mass conversion in each mode was established by analysing a m ixture of p rotein and peptide standards as described i n Table 5.
MALDI -TOF/MS data were acquired in either the positive reflectron or positive linear of operation with the accelerating voltage i n the ion source set to 1 5 KV. Once acquired the data was processed with the background subtraction (parameters: polyn umera l = 1 5, % below curve = 1 0) algorithms included with Masslyn x ™ .
MALDI-TOF/MS samples were prepared with a-cyano-4-hydroxycinnamic acid (HCCA, obtained from Sigma-Aidrich) as the m atrix. Satu rated m atrix solutions ( -1 0 mg/m l HCCA) were prepared as a 1 : 1 sol ution of acetonitrile and M i l li-Q® water with 0 . 1 %(v/v) TFA; this solution was mixed with equal volumes of the sam ple
solution before an aliquot was a pplied to the MALDI -TOF/MS sample plate and allowed to air-dry before being introduced into the m ass spectrometer.
Where indicated in Chapter 5, protein and peptide sam ples were de-salted by one of two m ethods : ( 1 ) prior to m ixing with the saturated m atrix solution, the sam ple was de-salted by passage through Millipore® Ziptip® 1J-C 1 8 pipette tips used according to the manufacturer's i nstructions. (2) The crystalline sam ple spots were prepared on the MALDI -TOF/MS sam ple plate using the fast evaporation m ethod of Vorm et a/ ( 1 994). The water-soluble contami nants within the crystals were removed by treating the spots with cold M illi-Q® water as described by Beavis and C hait ( 1 990) .
TABLE 5: STANDARDS USED TO CALIBRATE THE M@LDI LR MASS SPECTROMETER. Protein/Peptide standard Monoisotopic Molecular mass Average Molecular mass
(used for Reflectron mode calibration) (used for Linear mode calibration)
Adrenocorticotropic hormone 2,465. 1 99 Da 2,466.7 Da
fragment (residues 1 8-39)
Angiotensinogen (Renin substrate) 1 , 758.934 Da 1 ,760.0 Da
Angiotensin 11 1 ,046.542 Da 1 047.2 Da
Bovine pancreatic insulin 5730.61 Da 5,734.6 Da
Cytochrome C Not used 1 2,385 Da
2.4.7.2 ANALYSING RNASE A.
One millig ram of Bovine pancreatic ribonuclease Type I l l-A (Sigma-Aidrich®) was dissolved in 50 IJL in g uanidine-citrate buffer (section 2.4.7. 1 ) before 0.5 IJL of 1 M TCEP was added . TCEP was allowed to partially reduce the cysteines i n RNase A for 1 5 min at 25 °C before the immediate addition of 40 IJ L of a 0.5 M CDAP dissolved in 0. 1 M citrate buffer. The reaction solution was incubated with CDAP for a further 30 min at 25 °C before the reduced and cyanylated RNase A species were resolved by RP-H PLC.
Reaction solutions containing reduced and cya nylated isomers of RNase A were applied di rectly to a Amersham® IJ RPC C2/C 1 8 ST 4 . 6/1 00 RP-HPLC col u m n protected with t h e Jupiter™ 50 mm x 1 0 m m guard column (see Table 4 for details), a nd resolved using the RP-H PLC gradient profile descri bed by Figure 1 6. Each protei n fraction was col lected m anually as it eluted from the column.
The ammonia-catalysed S-cyanylated peptide cleavage reaction was carried out by adding 8 IJ L of a m monium hyd roxide (prepared as described in section 2.4.7. 1 ) and allowing it to react at 25 oc for one hour. The removal of ammonia, the final reduction of disu lfide bonds, a nd the subseq uent analysis of the peptide mixture by LC-ES I/MS and MALDI-TOF/MS was performed as described in section 2.4.7. 1 .
Methods and Materials.
FIGURE 16: RP-HPLC MOBILE PHASE GRADIENT FOR FRACTIONATING REDUCED AND CYANYLATED RNASE A ISOMERS WITH THE p.RPC ST 4.6/100 COLUMN.
CHEMSTA TION PROGRAM DETAILS G RADIENT PROFILE2
Time of each
Programmed components in the RP-HPLC Gradient Program ROW_H3.m Gradient Column ftush
m obile phase 1 100 Change. • % C % 0
�
e:. ·-· o min 5 75 20 70 c a.> > 5 n7II1 5 75 20 0 • acetonitrilefTFA (f) 50 -•-waterfTFA ... 0 6o min 5 65 30 c 40 .Q "§ 30 65 min 5 0 95 c a.> 20 • u c 70 min 5 75 20 u 0 10 0Flow Rate Max Pressure Limit 0 10 20 30 40 50 60 70
1. 0 mL/min 300 bar Retention Time (min)
1 Solvent A = 1 00% HPLC-grade methanol. Solvent C = 0. 1 %(v/v) TFA in fi�er sterilised Milli-Q water. Solvent D = 0.1 %(v/v) in HPLC grade acetonitrile. '%A,' '%C,' and '%0' refer to the concentration of each component as a percentage of the total mobile phase. 2 The gradient profile depicts the RP-HPLC mobile phase gradient resulting from the ChemStation program. This program was used exclusively with the �RPC ST 4.6/ 1 00 column described in Table 4.