Organelle Proteomics by Label-Free and SILAC-Based Protein Correlation Profiling - LC-MS/MS in Proteomics: Methods and Applications - page 254

L-glutamine (200 mM stock solution) (Gibco-Invitrogen);
and penicillin/streptomycin (10,000 U/10,000 μ gstock

solution).
4. Trypsin-EDTA solution (trypsin 200 mg/L and versene-
EDTA 500 mg/L) (Gibco-Invitrogen).
5. Sterile phosphate-buffered saline (PBS) (Cambrex Bio Sci-
ence, Copenhagen ApS, Denmark).
6. Filter units, MF75TM series (Nalge Nunc International,
NY).
7. Lysis buffer: 10 mM Tris-HCl (pH

= 7.5), 150 mM NaCl,

-mercaptoethanol, 1 mM MgCl 2 ,pro-
tease inhibitors, EDTA-free (CompleteTM tablets, Roche
Diagnostics, Mannheim, Germany). Must be made fresh.
8. Dounce tissue grinders with “tight” pestles. Depending
on the sample amount 1 and 7 mL grinders are generally
used.

0.5 NP-40, 0.1% β

1. Iodixanol OptiPrep R density gradient medium (Sigma-
Aldrich) or sucrose solution.
2. Dithiothreitol (DTT) (Sigma-Aldrich).
3. Iodoacetamide (Sigma-Aldrich).
4. SDS loading buffer.
5. In the case of in-solution digestion samples are dissolved in
6 M urea/2 M thiourea.
6. NuPAGE R Novex 4-12% Bis-Tris gel system (Invitrogen).
7. MOPS running buffer (Invitrogen).
8. Colloidal Blue Stain (Invitrogen).
9. Sequence grade-modified trypsin (Promega, Madison, WI)
( 15 ) .

2.2.Cell
FractionationandGel
Electrophoresis

2.3.Liquid
Chromatography
(LC)-MSandData
Analysis

1. Hydrophilic buffer A, containing 0.5% acetic acid (Sigma-
Aldrich) in water
2. Hydrophobic buffer B, containing 0.5% acetic acid in 80%
acetonitrile (Fisher Scientific)
3. STAGE tip for peptide purification ( 16 )
4. Reversed-phase chromatography columns (75 m ID) filled

with Reprosil C18-AQ, 3 mm beads HPLC bulk packing
material (Dr Maisch)
5. Protein identification software tools

μ

(such as Mascot

(MatrixScience)
6. Protein quantitation software (such as MSQuant, http://
msquant.sourceforge.net

or MaxQuant, http://www.

maxquant.org /( 17 ) .