After drying in a beckman berkeley ca paragon dryer

After drying in a Beckman (Berkeley, CA) Paragon dryer for 60 min, the gel is
stained for 10 min in a solution containing 0.2% bromophenol blue + 30% etha-
nol + 5% glacial acetic acid. The destaining solution consists of 30% ethanol +
5% glacial acetic acid solution.
4. The formation and stability of the HbSNO can confirmed by mass spectrometry.
In physiological phosphate buffer (pH 7.4) at 25 ° C, mass spectrometric analysis
shows that the half-life of HbSNO is about 24 h. The inherent stability of HbSNO,
and in general of protein RSNOs, is quite remarkable and contrasts strikingly
with that of low molecular weight RSNOs, which are exceedingly unstable under
physiological conditions (9 , 10) .
5. iNOS is responsible for the biosynthesis of nitric oxide from
L
-arginine. iNOS is
a soluble enzyme found in a variety of tissues/cell types including macrophages,
hepatocytes, and vascular smooth muscle (27) . iNOS activity is essentially inde-

Preparation and MS Analysis of S -Nitrosohemoglobin 163
pendent of calcium and has a K
m
of 16 µ M for
L
-arginine. Recombinant mouse
macrophage iNOS, stored frozen at –80 ° C, in 50 m M HEPES (pH 7.4) contain-
ing 10% glycerol, is stable for at least six months, but loses approximately
10–15% of its activity during a single freeze/thaw cycle. In the event that only a
portion of the enzyme is to be used in a single experiment, it is recommended that
the enzyme be aliquoted into smaller sizes and frozen at –80 ° C. iNOS is a
relatively unstable enzyme. Hence, to prevent loss of activity, keep the stock vial
of the enzyme on ice (0–4 ° C) at all times. The enzyme should be added to the
incubation medium (assay mixture) just before the start of the experiment.
6. The performance of the protein/peptide identification system is assessed in
advance by the use of protein/peptide calibration samples. Different amounts of
purified human hemoglobin sample, ranging from 100 to 300 ng as determined
by amino acid composition analysis, are digested with pepsin or endoproteinase
Glu-C; the peptide mixtures are then separated by RP-HPLC and detected on-line
by UV absorbance and ESI-MS. The reliability of the results obtained is dis-
cussed in Notes 7 and 8 .
7. UV absorbance detection. In general agreement with our previous experience
(15) , use of neither the chemical nor the enzymatic synthesis protocol is found to
interfere significantly with the UV absorbance detection of proteins and pep-
tides. A typical result is shown in Fig. 4 . After elution of the large injection peak,
the peptides derived from the digestion of 300 ng of HbSNO are detected as a
smooth baseline with a sensitivity limit in the picomole range and a typical peak
width of 30–40 s. Although not essential for protein identification, the UV chro-
matogram provides valuable information on the quality of the sample analyzed
and the level of contaminants and is useful for trouble-shooting the HPLC sys-
tem. The use of a microbore on-column detection system, low ID fused-silica
capillaries, and PEEK tubing minimizes the loss of chromatographic resolution
between the flow cell and the ion source of the mass spectrometer.