Supporting Information Methods S1
Purification of S-acylated proteins for proteomic analysis
The microsomal fraction of cell cultures was prepared as described previously (1). Microsomal protein (5
mg) was solubilised and free cysteines blocked by mixing for 12 h at 4°C in 5 ml total PBS containing
0.5% SDS, 1% Triton X-100 (Sigma-Aldrich), 0.5% Saponin (Calbiochem), protease inhibitors (Calbiochem
cocktail VI), 5 mM EDTA and 25 mM NEM (Thermo scientific). Proteins were precipitated using
chloroform/methanol (2), air dried, resuspended in 1 ml of resuspension buffer (6 M urea, 2% SDS in
PBS pH 7.4) by water bath sonication and gentle mixing. Half of each sample was combined with 3 ml
1M hydroxylamine (pH 7.4 with NaOH), 1 ml PBS, protease inhibitors, 5 mM EDTA and 500 µl of 3 mM
Biotin-HPDP (Thermo Scientific) in DMF and incubated at 25°C for 1 h with gentle mixing. The remaining
fraction was treated identically except that hydroxylamine was replaced with water. Proteins were
precipitated and resuspended as before. Resuspended samples were dissolved in 4 ml PBS, 5 mM EDTA,
0.1% Triton X-100 and protease inhibitors. Samples were centrifuged at 10 000 g at room temperature
for 10 min to remove particulates. Each sample was mixed with 100 µl of high capacity neutravidin
beads (Thermo scientific) for 1 hour at room temperature. Beads were recovered on 10 ml disposable
columns. Beads were washed three times with 5 ml PBS containing 0.2% SDS, 0.2% Triton X-100 and 500
mM NaCL. Columns were capped and proteins eluted with 500 µl of 80°C PBS containing 0.1% SDS, 0.1%
Triton X-100 and 1% 2-mercaptoethanol and incubated at 80°C for 15 min with vigorous shaking. The
eluate was collected by centrifugation of the column at 3000 g for 1 min. The elution process was
repeated with a further 500 µl of elution buffer and the eluates pooled and chloroform/methanol
precipitated.
Protein identification and quantitation
100 µg of trypsinised samples were labelled with iTRAQ reagents and pooled (3). Peptide samples were
separated by strong cation exchange (SCX) and fractions analysed using an ABI Q-star LC-MS/MS (4).
MS/MS spectra were searched against the TAIR9 Arabidopsis protein database using Mascot (v2.2)
(modifications: Fixed, iTRAQ four-plex (Lys), iTRAQ four-plex (N-terminal), NEM (Cys). Variable; oxidation
(Met), iTRAQ four-plex (Tyr); tolerances, MS – 0.2 kDa, MS/MS – 0.5kDa). The peptide-spectrum matches
from this search were analysed by Mascot Percolator (5) to calculate the false discovery rate scores (qvalue). Matches were then paired with iTRAQ reporter ion tag data using iSPY, an in-house updated
software expanding the capabilities of iTRACKER, which is freely available on request from KS Lilley
(Cambridge Centre for Proteomics, University of Cambridge, UK). A false discovery rate threshold of 1%
(q <= 0.01) was applied at the peptide level. For quantitation, the minimum reporter ion count was 15 in
one or more of the 4 reporter ions. Individual peptide levels were log. transformed and mean absolute
deviation normalized. Protein abundance was then calculated by averaging the abundance of all
detected peptides. Peptides were quantified only if they were unique to a single protein. All results
above these thresholds are reported in the relevant Supporting Information tables. Channels where
reported quantification was 0 were given a value of 0.00001 to allow relative quantification calculations
to be performed. Each experiment measured protein acylation from both WT and tip1-2 samples and
was repeated twice.
Data analysis
924 proteins were identified and constitute the raw data shown in Table S1. From these proteins a
candidate S-acylated protein list was generated. For a protein to be considered S-acylated it should be
enriched in the Hyd+ sample compared to the Hyd- (non-specific binding and thioester cleavage control)
sample. Proteins with a WT Hyd+ / WT Hyd- ratio less than 1 in either experiment were therefore
removed. The proteins excluded by these processes are listed in Table S2. The remaining proteins were
split into 3 groups based on the number of peptides used to identify them; high confidence of protein
identity (3 peptides or more in at least one experiment), medium confidence of protein identity (2
peptides in at least one experiment) and lower confidence of protein identity (one peptide only in either
or both experiments). Within each group proteins were given an enrichment rank order based on their
Hyd+/Hyd- ratio across all 4 potential observations of that protein to indicate confidence in S-acylation.
These putative S-acylated proteins are shown in Table S3. Common fortuitous proteomic contaminants
(e.g; ribosomal proteins) (3) were removed and are shown in Table S4. Proteins known to contain
moieties linked by thioester bonds not involved in the membrane association of proteins (e.g. nitrilase,
ubiquitin conjugating and ligating enzymes, various lipid synthesis enzymes, aminoacyl tRNA ligases)
were removed to Table S5 as the hydroxylamine / biotinylation method will also isolate these. The
presence and enrichment of these proteins in the hydroxylamine treated (Hyd+) samples provides
evidence that our method is capable of enriching for and identifying proteins containing thioester
bonds. The curated set of putative S-acylated proteins are shown in Table S6.
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