Protocol for Quantitative Proteomics of Cellular Membranes and Membrane Rafts - LC-MS/MS in Proteomics: Methods and Applications - page 234

as an unresolved band on a 1D SDS-PAGE gel ( 6 ) . Samples were
solubilised effectively in strong detergent and concentrated at the
interface of the 4% acrylamide stacking gel and the 20% acry-
lamide resolving gel. Each membrane protein sample for compar-
ison by quantitative proteomics was then confidently excised as a
single band, digested in-gel, and labelled with iTRAQ R reagents
for quantitative 2D LC-MS/MS (see Fig. 14.1 for workflow
scheme). Quantitative comparison of samples processed by this
gel-based method versus a typical in-solution digestion method
revealed a sixfold improvement in the MS ion intensity indicat-
ing greatly improved analytical sensitivity. This chapter describes
in detail the preparation of membrane and membrane raft sam-
ples, confirmation of the integrity of membrane raft isolation by
western blot analysis for the raft marker flotillin-1, isolation of the
membrane/membrane raft samples as an unresolved band on gel,
followed by in-gel digestion, strong cation exchange (SCX) frac-
tionation, and sample desalting in preparation for LC-MS/MS
analysis.

2. Materials

2.1. Isolationof
NeuronalPlasma
Membranes

1. Plasma membrane cell lysis buffer: 10 mM Tris-HCl, pH
7.4, 10 mM NaCl, 3 mM MgCl 2, ,10mMNaF,2mM
Na 3 VO 4 , 1 mM EGTA, 1 mM EDTA, 0.2 mM PMSF,
protease inhibitor cocktail (Sigma, Gillingham, UK; see

Note 1 ).
2. Membrane solubilisation buffer: 10 mM Tris-HCl, pH 7.4,
10 mM NaCl, 3 mMMgCl 2, ,10mMNaF,2mMNa 3 VO 4 ,
1mMEGTA,1mMEDTA,0.2mMPMSF,0.1%SDS
(w/v).
3. Tris-buffered saline (TBS): Prepare a 10X stock of TBS
(250 mM Tris-HCl, pH 8.0, 1.4 M NaCl, 50 mM KCl)
and store at 4 ◦ C. As required, dilute 100 mL of 10X stock
with 900 mL of water prior to use.
4. Mini glass homogenisers, 1 mL capacity (Jencons, Leighton
Buzzard, UK).

2.2. Isolationof
NeuronalMembrane
Rafts

1. MES/NaCl buffer: 25 mM MES, 150 mM NaCl, pH 6.5.
Store at 4 ◦ C.
2. MES buffer: 25 mM MES, pH 6.5. Store at 4 ◦ C.
3. Membrane raft cell lysis buffer: 25 mM MES, 150 mM
NaCl, 10 mM MgCl 2 ,10mMNaF,2mMNa 3 VO 4 ,1mM
EGTA, 5 mM DTT, 0.2 mM PMSF pH 6.5 ( see Note 2 ),