Proteomic Studies of Isolated Lipid Droplets from Bacteria, C. elegans, and Mammals - Methods in Cell Biology - page 8

3.3 The sample is alkylated for 45 min by adding 55 mM iodoacetamide in
the absence of light.
3.4 Proteins are then incubated with trypsin solution (10 ng/

m lin25mM

ammonium bicarbonate) for 30 min on ice.
3.5 After removing excess enzyme solution, 25 mM ammonium
bicarbonate is added.
3.6 Total proteins/proteins in-gel are digested at 37 C overnight.
3.7 5% formic acid is added to stop the digestion reaction.
3.8 The sample solution is then vortexed and centrifuged, and the
supernatant is collected.
3.9 The gel pieces are further extracted using 60% acetonitrile in 1% formic
acid twice.
3.10 The extracts and the supernatant are combined and dried under
vacuum to get the tryptic peptides mixture.
3.11 The peptides mixture is dissolved in 1% formic acid and then applied
to a C18 trap column.
3.12 The eluted peptide is subjected to nano-LC-ESI-LTQ Orbitrap XL
MS/MS analysis.
3.13 The Orbitrap mass spectrometer is set to operate under the data-
dependent mode and with an initial scan range of 300-1800 Da.
Step 4 MS data analysis and protein identification
All MS/MS data are searched against each individual database for bacteria, C.
elegans , and mammals, and thus parameters may vary. Furthermore, BioWorks (3.31
sp1) Sequest search parameters need to be set as follows: enzyme: trypsin; precursor
ion mass tolerance: 2.0 Da; and fragment ion mass tolerance: 1.0 Da. The variable
modification is set to oxidation of methionine at (Met

15.99 Da). The fixed
modification is set to carboxyamidomethylation of cysteine at (Cys

þ

57.02 Da). The
search results are filtered with Xcorr versus charge values of Xcorr (

þ

þ 2) 2.5 and

>

Xcorr ( þ 3) 3.5. Peptide mass accuracy is set at 5 ppm, SP score 500, RSp 5.

> < > <

Distinct peptides 2 and miss cleavages 2 ( Fig. 1.3 ).

Step 5 Verification of identified LD proteins
There are several ways to verify the MS-identified LD proteins. The following
are standard methods that have been developed.
5.1 Western blot is used to analyze the enrichment of proteins in the LD
fraction ( Fig. 1.4 A-D).
5.1.1 LD, TM, Cyto, and PNS are collected and proteins prepared as
described above.
5.1.2 Equal amount of proteins are separated by SDS-PAGE and
transferred to PVDF membrane.
5.1.3 Target proteins are identified using specific antibodies. LD
markers are applied as ADRP for mammals, DHS-3 for C.
elegans , MLDS for bacterium RHA 1.
5.2 GFP fusion protein is used to determine protein LD localization
( Fig. 1.4 E and F).