Biology Reference
In-Depth Information
Chapter 14
Combining Chymotrypsin/Trypsin Digestion to Identify
Hydrophobic Proteins from Oil Bodies
Martina Vermachova , Zita Purkrtova , Jiri Santrucek , Pascale Jolivet ,
Thierry Chardot , and Milan Kodicek
Abstract
Oil bodies, lipid-storage organelles, are stabilized by a number of specifi c proteins. These proteins are very
hydrophobic, which complicates their identifi cation by “classical” proteomic protocols using trypsin
digestion. Due to the lack of trypsin cleavage sites, the achievable protein coverage is limited or even insuf-
fi cient for reliable protein identifi cation. To identify such proteins and to enhance their coverage, we
introduced a modifi ed method comprising standard three-step procedure (SDS-PAGE, in-gel digestion,
and LC-MS/MS analysis). In this method, chymotrypsin, single or in combination with trypsin, was used,
which enabled to obtain proteolytic peptides from the hydrophobic regions and to identify new oil bodies'
proteins. Our method can be easily applied to identifi cation of other hydrophobic proteins.
Key words
Chymotrypsin, Hydrophobic proteins, In-gel digestion, LC-MS/MS, Oil bodies
Abbreviations
AA
Acrylamide
ACN
Acetonitrile
APS
Ammonium persulfate
BIS
-methylenebisacrylamide
CBB Coomassie Brilliant Blue
DTT Dithiothreitol
FA Formic acid
IAA Iodoacetamide
IB Isolation buffer
OBs Oil bodies
PLB Protein loading buffer
SDS Sodium dodecyl sulfate
TEMED
N
,
N
,
N
N,N
′
′
,
N
′
-tetramethylethylenediamine