Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
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Figure 1. ESI-MS spectrum showing the selective reaction of ebselen with cysteine in the presence of methionine, tryptophan, and valine. The<br />
insets show the agreement of the simulated product peak isotopic distribution (top) with the recorded reaction product ion of m/z 397 (bottom).<br />
some cases) at room temperature. On the basis of these revealed<br />
features of the reaction, associated analytical applications were<br />
explored, such as the selective identification of thiol-containing<br />
peptides from mixtures and measurement of the number of<br />
cysteine residues of proteins. In addition, the dissociation behaviors<br />
of the derivatized protein/peptide ions were also examined.<br />
EXPERIMENTAL SECTION<br />
Electrospray ionization (ESI) of samples was performed using<br />
a Thermo Finnigan LCQ DECA Mass Spectrometer (San Jose,<br />
CA) or a hybrid triple-quadrupole-linear ion trap mass spectrometer<br />
(Q-trap 2000; Applied Biosystems/MDS SCIEX, Concord,<br />
Canada). The sample injection flow rate was 10 µL/min. A high<br />
voltage of +5 kV was applied to the spray probe for the positive<br />
ion mode. For the Thermo Finnigan LCQ DECA mass spectrometer,<br />
the optimized heated transfer capillary tube temperature was<br />
150 °C. Collision-induced dissociation (CID) was used for further<br />
structural confirmation of the product ions. Data acquisition was<br />
performed using Xcalibur (rev. 2.0.7, Thermo Scientific, San Jose,<br />
CA). For the Q-trap 2000 mass spectrometer, the mass spectrometer<br />
curtain gas (N 2) was kept as 20 (manufacturer’s units) and<br />
the declustering potential was set at 10 V. Collision induced<br />
dissociation (CID) was carried out to provide ion structural<br />
information using enhanced product ion scan mode. Precursor<br />
ion scanning (PIS) was used to select reaction products by<br />
monitoring the characteristic fragment ions, and N2 was used<br />
as the collision gas. Data acquisition was performed using the<br />
Analyst software (version 1.4.2, Applied Biosystems/MDS<br />
SCIEX, Concord, Canada). Deconvolution of mass spectra was<br />
carried out using Mag-Tran 1.03b2 software (Amgen Inc.,<br />
Thousand Oaks, CA) written based on the ZScore algorithm. 40<br />
High-Resolution MS. High-resolution LTQ-Orbitrap mass<br />
spectrometer (Thermo Electron, Bremen, Germany) with a<br />
(40) Zhang, Z.; Marshall, A. J. Am. Soc. Mass Spectrom. 1998, 9, 225–233.<br />
6928 <strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010<br />
modified nanoelectrospray ionization (nano-ESI) was used for<br />
collecting high-resolution data. The system was operated in the<br />
positive ion mode with a resolving power of 60 000 at m/z 400.<br />
The Molecular Weight Calculator (http://ncrr.pnl.gov/software/)<br />
was used to simulate the isotope distribution for thiol-derivatized<br />
products.<br />
<strong>Chemical</strong>s. The 20 amino acids, angiotensin II human (FW<br />
1046 Da), bradykinin acetate (FW 1060 Da), MRFA (Met-Arg-<br />
Phe-Ala) acetate salt (FW 523 Da), �-lactoglobulin A from bovine<br />
milk, tris(2-carboxyethyl)phos-phine hydrochloride (TCEP), DLdithiothreitol<br />
(DTT), TPCK-treated trypsin from bovine pancreas<br />
(MW ∼23.8 KDa), ammonium bicarbonate, 1,4-benzoquinone, and<br />
N-(phenylseleno)phthalimide were purchased from Sigma-Aldrich<br />
(St. Louis, MO). Glutathione (GSH) (reduced form, MW 307 Da)<br />
and ebselen were obtained from TCI America (Tokyo, Japan) and<br />
Calbiochem (Cincinnati, OH), respectively. HPLC-grade methanol<br />
and acetonitrile from GFS <strong>Chemical</strong>s (Columbus, OH) and Sigma-<br />
Aldrich (St. Louis, MO) were used, and acetic acid was purchased<br />
from Fisher Scientific (Pittsburgh, PA). The deionized water used<br />
for sample preparation was obtained using a Nanopure Diamond<br />
Barnstead purification system (Barnstead International, Dubuque,<br />
IA).<br />
Protein Reduction. A volume of 175 µL of 0.1 mM �-lactoglobulin<br />
A in methanol/water (1:1 by volume) containing 2% acetic<br />
acid and 17.5 µL of 50 mM TCEP in 20 mM ammonium<br />
bicarbonate aqueous solution were mixed resulting in the molar<br />
ratio of 1:50 (protein/TCEP). The protein was reduced by TCEP<br />
for 3.5 h at room temperature. Then Millipore-ZipTip Pipette Tips<br />
were used to remove TCEP and ammonium bicarbonate via<br />
desalting.<br />
RESULTS AND DISCUSSION<br />
Reaction Selectivity. In this experiment, the selectivity of the<br />
derivatization reaction was examined first using amino acids as