Biology Reference
In-Depth Information
the proteins before digestion is a commonly used strategy for
reducing the complexity of the sample, increasing the total num-
ber of analyzed peptides after the combination of the different
fractions which usually lead to the identifi cation of large number
of proteins [
3
].
In laboratory practice, the use of mass spectrometry means
that all of the work should be done in a very clean environment,
and mass-spectrometry compatible buffers and reagents should be
used during protein extraction, fractionation and digestion. One of
the classical drawbacks of using standard protocols is the poor
identifi cation rate of membrane and cell-wall proteins. The solubi-
lization of these proteins requires a high concentration of deter-
gents, not only during the initial steps of the extraction but also for
the solubilization of the purifi ed protein pellet. There are systems
for removing detergents such as SDS from the sample; they are
expensive and not effective in all of the situations. Another prob-
lematic step is the protein fractionation. Despite the fact that frac-
tionation protocols are not very complex (classically done by
column chromatography, i.e., FPLC), they are time consuming
and require large amounts of proteins.
In this chapter we show a complete LC-MS/MS proteomics
workfl ow, fully compatible with high concentrations of SDS. We
implemented a rapid standard SDS-PAGE step that is used for pro-
tein pre-fractionation, washing (removal of detergents and other
contaminants), and a high performance in-gel digestion of proteins.
This workfl ow is time effective and can be completed in less than 3
days (day 1, protein extraction and quantitation; day 2, protein frac-
tionation and digestion; day 3, peptide desalting and LC-MS/MS
analysis). Peptides are resolved and identifi ed using an nano HPLC
system coupled to a LTQ-Orbitrap mass spectrometer. In our hand
this protocol leads to a reproducible identifi cation of more than
1,500 proteins starting from 50
g of total protein in many differ-
ent biological systems from microbes to plants.
μ
2
Materials
Prepare all solutions using ultrapure water (bidistilled, deionized)
and analytical/ultra HPLC grade reagents.
2.1 Buffers
for Protein Extraction
and Quantitation
1. Extraction buffer: 100 mM Tris-HCl, pH 8.0, 10 % (v/v)
Glycerol, 2 mM PMSF, 10 mM DTT, 1.2 % (v/v) Plant prote-
ase inhibitor cocktail (Sigma P9599). Prepare fresh.
2. Buffer RS: 8 M Urea, 4 (w/v) % SDS. Buffer RS can be stored
for 1 year at room temperature.
3. BCA Reagent A: sodium bicinchoninate (0.1 g), Na
2
CO
3
H
2
O
(2.0 g), sodium tartrate (dihydrate) (0.16 g), NaOH (0.4 g),
⋅
