176 T. Oe et al.
The mass spectrometer was equipped with a nitrogen laser
(337 nm, 3 ns pulse width, 20 Hz repetition rate) and had a
flight path of 200 cm. All spectra presented were acquired in
positive-ion mode in reflection mode with the following
parameters. For PTH-amino acids: accelerating voltage,
10 kV; grid voltage, 70%; extraction delay time, 100 ns; low
mass gate, 100 Da; shots in the spectrum, 100–150. For tryptic
peptides: accelerating voltage, 20 kV; grid voltage, 64%;
extraction delay time, 100 ns; low mass gate, off; shots in the
spectrum, 100. TOF MS experiments were performed in the
range of m/z 100–1500 for PTH-amino acids and 500–5000 for
tryptic peptides, respectively. All acquired data were
processed using Data Explorer version 4.0.0.0.
following criteria: curve, linear; origin, ignored. The above
experiment was performed in triplicate (n ¼ 3).
Synthesis of [13C6]-PITC
The isothiocyanate group was constructed by the method
described by Bures et al.27 with minor modifications. [13C6]-
Aniline (250 mg, 2.5 mmol) in CHCl3 (2 mL) was allowed to
react with O,O0-di-2-pyridyl thiocarbonate (1.25 g, 5 mmol) at
room temperature for 3 h then at 508C for 1 h. After the
reaction, the solvent was evaporated and the residue
was applied to a flash silica gel chromatography column
with n-hexane to remove excess reagent. The eluate was
then evaporated to give [13C6]-PITC as a colorless oil
(392 mg, quant.). High-resolution FABMS calculated for
13C162C1H5N1S1: theoretical, m/z 141.0344; found, m/z 141.0324
Instrumental analyses
1H-NMR
.
(Mþ ). 1H-NMR (500 MHz, CDCl3, d): 7.05–7.25 and 7.35–7.55
(doublet of multiplets, J(13CꢁH) ¼ 167 Hz, HAr, 5H). IR (KBr,
NMR spectra were recorded at 258C at 500 MHz using an
ECP-500 (JEOL Ltd., Tokyo, Japan) equipped with a 3 mm
indirect detection gradient probe (Wilmad Glass Co. Inc.,
Buena, NJ, USA). [13C6]-PITC (ca. 1 mg) was dissolved in
150 mL of CDCl3 (containing 0.03% TMS). Data processing
was conducted directly on the NMR spectrometer. Chemical
shifts are reported on the d scale (ppm) by assigning the TMS
peak.
cmꢁ1): 2078 (vs, NCS).
Synthesis of [13C6]-PTH-Asp
Ang II (7 mM in 0.1 M HCl, 10 mL ꢀ 10) solvent was
evaporated under a N2 stream. The residue was allowed
to react with 10 mL of [13C6]-PITC solution ([13C6]-PITC/
triethylamine/water/ethanol 1:1:1:7, v/v/v/v) at 558C for
15 min on a heating block. The reaction mixture was dried
under a N2 stream and the residue was then washed with n-
hexane/ethyl acetate (15:1, v/v, 200 mL) and ethyl acetate
(200 mL). Ethylmercaptan in TFA (0.1%, v/v, 20 mL) was
added to the tube at 558C for 15 min on a heating block. After
evaporating the solvent under a N2 stream, the residue was
redissolved in water (20 mL) at 558C for 5 min and 50% (v/v)
aqueous TFA (20 mL) at 558C for 60 min. The reaction mixture
was dried under a N2 stream and the residue was then
dissolved in 50% (v/v) ethanol (50 mL). The solution from
each tube was then applied to HPLC system 2, the fraction
between tR 4.3 and 5.3 min was collected, dried under a N2
stream, and redissolved in 50% (v/v) aqueous ethanol (7 mL)
as [13C6]-PTH-Asp stock solution (100 mM). MALDI-TOF MS
(DHB): m/z 257.07 (MHþ).
High-resolution FABMS
High-resolution mass spectra were recorded using a JMS-700
double-focusing magnetic sector mass spectrometer (JEOL
Ltd., Tokyo, Japan).
IR
IR spectra were determined at ambient temperature using an
FTIR-400 (JASCO Co., Tokyo, Japan). [13C6]-PITC was
diluted with CHCl3. An aliquot of the solution was dropped
on a KBr disk and analyzed after drying.
Edman degradation reaction for BSA
Each BSA solution (20, 50, 100, 200, 500, 1000, 2000, 5000,
10000, 20000, and 50000 pmol/10 mL in 0.1 M HCl) was
transferred to a microcentrifuge tube and evaporated under
a N2 stream using a Jet Air vaporizer (Ishii Shoten Co., Tokyo,
Japan). Each residue was allowed to react with 10 mL of PITC
solution (PITC/triethylamine/water/ethanol 1:1:1:7, v/v/
v/v) at 558C for 15 min on a heating block (Yamato Scientific
Co., Ltd., Tokyo, Japan). The reaction mixture was evapor-
ated under a N2 stream and the residue was then washed
with n-hexane/ethyl acetate (15:1, v/v, 200 mL), ethyl acetate
(200 mL), and acetone (200 mL ꢀ 3). Ethylmercaptan in TFA
(0.1%, v/v, 20 mL) was added to the tube at 558C for 15 min on
a heating block. After evaporation under a N2 stream, the
residue was redissolved in 25% (v/v) aqueous TFA (20 mL)
and heated at 658C for 30 min. At that stage, an aliquot of this
solution can be used for further protein identification if
required. PTH-nor-Val (1 nmol/100 mL of 50% aqueous
ethanol, v/v) was added to each tube as an IS. An aliquot
(5 mL) of the final solution was analyzed using HPLC system
1. The peak area ratio (PTH-Asp/PTH-nor-Val) was then
plotted against BSA concentration. The calculations were
performed using Microsoft Office Excel 97–2003 with the
Calibration curve using PTH-Asp and
[13C6]-PTH-Asp for BSA quantitation
The calibration curve was prepared with PTH-Asp standard
and [13C6]-PTH-Asp. PTH-Asp solution (10, 20, 50, 100, 200,
500, or 1000 pmol/10 mL of 50% aqueous ethanol, v/v) and
[13C6]-PTH-Asp solution (1000 pmol/10 mL of 50% aqueous
ethanol, v/v) were mixed. BSA (1 nmol/10 mL in 0.1 M HCl)
was allowed to react with Edman reagent as described in the
HPLC-UV experiment and [13C6]-PTH-Asp (1000 pmol/
10 mL of 50% aqueous ethanol, v/v) was added as an IS.
For MALDI-TOF MS analyses, aliquots (0.5 mL) were loaded
on the MALDI sample plate followed by 0.5 mL of 100 mM
DHB in a mixture of water/acetonitrile/TFA (50:50:0.1, v/v/
v) containing internal calibrants (Arg, 10 pmol, MHþ
175.1195; Trp, 10 pmol, MHþ 205.0977; and Ang IV 1 pmol,
MHþ 775.4143) and allowed to dry at room temperature.
After the MALDI-TOF MS analyses, the peak area ratios of
PTH-Asp/[13C6]-PTH-Asp (monoisotopic MHþ; intensity @
251/intensity @ 257) were plotted against PTH-Asp concen-
trations. The calculations were performed using Microsoft
Copyright # 2009 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 173–179
DOI: 10.1002/rcm