11. The obtained peptide extract should be used for mass spectrometric analysis. Samples can be stored at À20
C for a week or
at À80
C for a month.
3.5 Tandem Mass
Spectrometric
Analysis Combined
with Nano-Liquid
Chromatography
1. Centrifuge samples at 3,500 Â g for 1 min at 20
C.
2. Ultrasonicate (20 W; 30 kHz) samples for 10 min.
3. Centrifuge samples at 3,00 Â g for 10 min at 20
C (to precipitate the smallest pieces of gel or other possible mechanical
contaminants).
4. Transfer the supernatant carefully so as not to touch the possible sediment (sometimes it can be difficult to see with the
naked eye) into clear glass or polypropylene inserts for vials.
5. Place the inserts with samples to vials and close with the Snap-It
Caps and Septa. Vials are loaded to the autosampler.
6. Use HPLC (liquid chromatography) system in column-prep
column configuration.
7. Apply samples to the Acclaim PepMap (2 cm  75 μm, C18,
3 μm, 100 A) pre-column at a flow rate of 2 μL/min for
10 min.
8. Separate samples on the Acclaim PepMap (15 cm  75 μm,
C18, 3 μm, 100 A) column at a flow rate of 300 nL/min in a
linear rising gradient of Buffer B between 5% and 40% for
120 min.
9. Regenerate the column and pre-column after each assay by
flushing 100% Buffer B for 7 min and then equilibrating it
with 5% Buffer B for 15 min.
10. To eliminate the possibility of incomplete washout through the
column and the pre-column between the samples, pass 2 short
(25 min) gradients from 5% to 95% of Buffer B with a 5-min
washout of 100% Buffer B at the end.
11. Apply a tryptic hydrolyzate solution of β-galactosidase (in an
aqueous solution containing 0.1% FA and 20 fM of
β-galactosidase hydrolyzate) using a gradient elution for
15 min (5–25% of system B) with the above-mentioned conditions of regeneration of the column and pre-column before
each stab of the sample.
12. Use β-galactosidase spectra for calibration and control of stability, productivity, and reproducibility of the mass
spectrometer.
13. Each sample is analyzed in three technical replicates for the Full
MS top 20 mass spectrometer to compile a database of identified proteins and to align the output time of individual peptide
components between experiments.
198
Julia Bespyatykh et al.
C for a week or
at À80
C for a month.
3.5 Tandem Mass
Spectrometric
Analysis Combined
with Nano-Liquid
Chromatography
1. Centrifuge samples at 3,500 Â g for 1 min at 20
C.
2. Ultrasonicate (20 W; 30 kHz) samples for 10 min.
3. Centrifuge samples at 3,00 Â g for 10 min at 20
C (to precipitate the smallest pieces of gel or other possible mechanical
contaminants).
4. Transfer the supernatant carefully so as not to touch the possible sediment (sometimes it can be difficult to see with the
naked eye) into clear glass or polypropylene inserts for vials.
5. Place the inserts with samples to vials and close with the Snap-It
Caps and Septa. Vials are loaded to the autosampler.
6. Use HPLC (liquid chromatography) system in column-prep
column configuration.
7. Apply samples to the Acclaim PepMap (2 cm  75 μm, C18,
3 μm, 100 A) pre-column at a flow rate of 2 μL/min for
10 min.
8. Separate samples on the Acclaim PepMap (15 cm  75 μm,
C18, 3 μm, 100 A) column at a flow rate of 300 nL/min in a
linear rising gradient of Buffer B between 5% and 40% for
120 min.
9. Regenerate the column and pre-column after each assay by
flushing 100% Buffer B for 7 min and then equilibrating it
with 5% Buffer B for 15 min.
10. To eliminate the possibility of incomplete washout through the
column and the pre-column between the samples, pass 2 short
(25 min) gradients from 5% to 95% of Buffer B with a 5-min
washout of 100% Buffer B at the end.
11. Apply a tryptic hydrolyzate solution of β-galactosidase (in an
aqueous solution containing 0.1% FA and 20 fM of
β-galactosidase hydrolyzate) using a gradient elution for
15 min (5–25% of system B) with the above-mentioned conditions of regeneration of the column and pre-column before
each stab of the sample.
12. Use β-galactosidase spectra for calibration and control of stability, productivity, and reproducibility of the mass
spectrometer.
13. Each sample is analyzed in three technical replicates for the Full
MS top 20 mass spectrometer to compile a database of identified proteins and to align the output time of individual peptide
components between experiments.
198
Julia Bespyatykh et al.