Biology Reference
In-Depth Information
an error tolerant search is performed automatically to the unassigned
spectra. When this results in a negative result, de novo sequencing is
automatically applied to the unassigned spectra. This de novo approach
is a crucial step since it allows a homology search of sequences instead
of a search based on
m
/
z
values. An
in silico
innovation in the pro-
posed workfl ow is the visualization of the peptides that were assigned
to specifi c proteins using Cytoscape. By a simultaneous visualization
of the signifi cant hits of the orphan specifi c search and the hits of the
cross-species search, the confi dence of the orphan specifi c hits increases
(Fig.
2
). Moreover, some peptides wrongly assigned to cross-species
hits are visualized and can be removed. Moreover, Cytoscape also
allows the visualization of the protein inference, a common problem
in a cross-species approach (Figs.
3
and
4
).
4
Materials (Peptide Based Approach)
4.1 Protein
Extraction
1. Liquid nitrogen.
2. Pestle and mortar (washed and baked in an oven at 180 °C
overnight prior to use).
3. Phenol, acetone and methanol compatible tubes and pipettes.
4. 10 % (w/v) DTT. Make freshly in MQ H
2
O.
5. Extraction buffer: 50 mM Tris-HCl, pH 8.5, 5 mM EDTA,
100 mM KCl, 1 % (w/v) DTT, 30 % (w/v) sucrose, and com-
plete protease inhibitor cocktail (according to the manufac-
turer, Roche Applied Science) in MQ H
2
O. Prepare freshly.
6. Tris buffered phenol, pH 8 (
see
Note 1
).
7. Precipitation solution: 100 mM ammonium acetate in metha-
nol. Store at −20 °C.
8. Cold acetone. Store at −20 °C.
9. Lysis solution: 8 M urea, 5 mM DTT.
10. Protein Quantifi cation kit 2D Quant (GEhealthcare, Diegem,
Belgium).
4.2 Protein Digestion
and Cleanup
1. 0.5 M Iodoacetamide (IAA).
2. 100 mM Ammonium bicarbonate (ABC).
3. 0.2
l Trypsin (Promega).
4. 10% trifl uoro acetic acid (TFA).
μ
g/
μ
5. Solid phase extraction (SPE) column (Supelco Inc, Bellefonte,
PA, USA).
6. Acetonitrile.
7. 0.1 M ammonium formate (AF).
8. SpeedVac.
9. NanoDrop (Thermo Scientifi c, Waltham, MA, USA).
