Biology Reference
In-Depth Information
Chapter 25
An Improved Detergent-Compatible Gel-Fractionation
LC-LTQ-Orbitrap-MS Workfl ow for Plant and Microbial
Proteomics
Luis Valledor and Wolfram Weckwerth
Abstract
In proteomics, liquid chromatography coupled to mass spectrometry (LC-MS/MS) is an invaluable
technique to accurately identify and quantify large sets of proteins. In this chapter we show a time-effective,
and detergent compatible, Gel-LC-LTQ-Orbitrap/MS proteomics workfl ow. The compatibility of this
protocol with high concentrations of detergents signifi cantly increases the extraction yield and the abundance
of membrane proteins while gel fractionation increases the number of protein identifi cations. In our hands
this workfl ow allows the identifi cation of more than 1,500 proteins per sample, harvesting less than 20 mg
of fresh weight material, in many different organisms such as
Chlamydomonas
,
Cyanothece
,
Arabidopsis
, or
Nicotiana
, various microbes and enriched microbial samples.
Key words
Green proteomics, LC-MS, Orbitrap, Detergent-compatible, Membrane proteins, Plant
Systems Biology
1
Introduction
Nowadays LC-MS/MS provides the highest resolution for the
identifi cation of peptides and the mapping of the proteomes [
1
].
Proteins are extracted and fractionated using standard protocols,
which have been adapted to be compatible with MS (reduced con-
centration of detergents and buffers), and then digested to tryptic
peptides by the use of endopeptidases. These tryptic peptides are
fi rst separated in a nanoHPLC system, generally coupled to a C18
column, and then identifi ed using tandem mass spectrometry [
2
].
This methodology give us the possibility of identify and accurately
quantify a huge number of proteins per run.
Even with the use of the next generation of mass spectrome-
ters, with an increased resolution, sensitivity, dynamic range, and
speed, the identifi cation of all the ions co-eluting from the col-
umn remains one of major bottlenecks. The pre-fractionation of
