Biology Reference
In-Depth Information
20 mM ammonium formate (pH 10). The Elution of the
peptides takes place in an excess of 0.1 % FA in water at a fl ow
rate of 20
l/min to reach a tenfold dilution before loading on
the analytical column.
3. Elute the peptide from the analytical column at 0.2
μ
l/min
using 0.1 % formic acid in water as eluent A and 0.1 % formic
acid in ACN as eluent B. The elution pattern is dependent on
the nature of the eluted sample from the fi rst column and is
controlled by a curve setting. The curve determines the rate at
which the solvent is to change to the new proportions and/or
fl ow rates. Curves are specifi ed by number with available choices
from 1 to 11. Curve 1 immediately goes to specifi ed conditions;
curves 2-5 are convex; curve 6 is linear; curves 7-10 are concave
and curve 11 maintains start condition until next step. All gradi-
ent slopes are linear (curve 6), except for the 10-40 % B step of
the 12 % ACN fractions (concave curve 7) and for the fractions
35 and 65 % ACN (convex curve 5). Separation should be car-
ried out using 5 % B for 1 min, 10 % B for 2 min, 10-40 % B
over 62 min, and 40-85 % B over 9 min. After 6 min of rinsing
with 85 % B and a linear gradient back to 5 % B over 2 min the
column is re-equilibrated at initial conditions. The analytical col-
umn temperature should be maintained at 35 °C.
μ
4. Perform the mass spectrometric analyses in positive mode using
ESI with a NanoLockSpray source. Spray the eluates immedi-
ately into the Q-TOF device with lock mass at a fl ow rate of
0.2
l/min. Set the lock mass channel at 30 s. Select for MS/
MS, the three most intensive multiply charged ions eluting
from the column for fragmentation. Detect the eluting peptide
ions in the MS survey scan (0.6 s) from m / z of 300 to 1,400.
Set a dynamic exclusion window was at 60 s ( see Note 7 ).
μ
5. Search the obtained peak lists against the species specifi c data-
base using Proteinlynx Global Server (PLGS 2.4, Waters) with
the following settings peptide tolerance 20 ppm, fragment tol-
erance 0.05 Da and one tryptic miscleavage,
Carbamidomethylation of C fi xed modifi cation, and oxidation
of M as variable modifi cation ( see Note 7 ).
6. Perform an error tolerant search (“automod” query) on the
spectra that did not result in an identifi cation in the non-error
tolerant query. Allow one nonspecifi c cleavage and one amino
acid substitution or modifi cation per peptide. Proteins identi-
fi ed with at least one peptide with a ladder score above 50 are
considered signifi cant.
7. Perform a de novo determination on the spectra that did not
result in an identifi cation “automod” query ( see Notes 8 and 9 ).
8. Export all the peptide tables of the different 1D fractions and
merge them into one Excel fi le 2010, i.e., original fi le.
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