S.1 Supplemental Material S.1.1. Preparation of Cell Lysates Reagents. Heavy isotope preparations of L-lysine (6C13 2N15, 608041) and L-arginine (6C13 4N15, 608033) were purchased from Sigma-Isotec. SILAC RPMI 1640 (89984) medium was purchased from Thermo Scientific. Natural isotope L-lysine (6C12 2N14, L5501 ), L-arginine (6C12 4N14, A5006) and L-proline (unlabeled, P5607) were purchased from Sigma. Dialyzed FBS (26400-044) was purchased from Invitrogen. NE-PER Nuclear and Cytoplasmic extraction reagents (78835) were purchased from Thermo Scientific. Complete Protease inhibitor cocktail was purchased from Roche (11697498001). Phosphatase inhibitor cocktail 2 was purchased from Sigma (P5726). Cell Culture. Bax+/- and Bax-/- HCT116 cell lines were a gift from B. Vogelstein. Jurkat cells were purchased from American Type Culture Collection. All cell lines for the SILAC experiment were cultured and expanded in SILAC RPMI 1640 supplemented with L-glutamine, L-proline and 10% dialyzed fetal bovine serum. The heavy isotope medium was supplemented with heavy isotope L-lysine at 50 ug/ml and heavy isotope L-arginine at 40 µg/ml. The light isotope medium was supplemented with both of these amino acids containing natural isotopes. Jurkats used for the chemical labeling experiment were cultured in standard RPMI1640 with 10% FBS. HCT116 cell lines were treated with Apo2L at 200 ng/ml ligand for 100 minutes at 37°C. Jurkat etoposide experiments were performed on 1 million cells/mL in suspension with 50 µM etoposide for 14 hours at 37°C.
Preparation of cell lysates. Lysates prepared for SILAC substrate
added phosphatase and protease inhibitors (see above). Cultured adherent cells were grown to 80% confluency and suspension cells were grown to a density of 1.0 x 106 cells per ml. Protein levels were quantitated by BCA, adjusted to equal concentrations and heavy and light isotope extracts were mixed equally, then stored at – 80 oC. Lysates
prepared
for
peptide
based
immunoaffinity
enrichment
(PTMScan): cells were cultured under the same conditions and densities as above and harvested and washed in PBS. Pellets were lysed in 20 mM HEPES pH 8.0, 9 M urea with phosphatase and protease inhibitors (as above). Lysates were sonicated with 2 x 30 second bursts at 30 watts then centrifuged at 15oC at 16,000 x g for 15 min. The cleared supernatant was harvested and stored at – 80 o
C.
S.1.2 Global Analyzer of SILAC-derived Substrates of Proteolysis (GASSP) S.1.2.1.SDS-PAGE: The Light (untreated) and Heavy (Apo2L treated) lysates (HCT 116 BAX /- or +/-) were mixed at 1:1 ratio and proteins separated on 4-12% Bis-Tris gel (Invitrogen, Carlsbad, CA) at 125 V for 40 min. Protein bands were visualized using Simply Blue staining solution (Invitrogen, Carlsbad) overnight. Destaining was performed in MQ water for 1h.
S.1.2.2. Tryptic Digestion: Entire lanes were excised and divided from top to bottom of the gel into 20 regions.
Gel
bands
were
further
destained
in
50
mM
ammonium
bicarbonate/50% acetonitrile (ACN) solution for 30 min followed by dehydration in 100% ACN for 10 min. Trypsin (Promega, Madison, WI) solution at 0.02 µg/µL
bicarbonate at 37°C overnight. Peptides were extracted with 50% ACN/0.1% trifluoroacetic acid (TFA) followed by 100% ACN. Extracts were combined and dried down completely. Peptides were then reconstituted in 2%ACN/0.1% formic acid (FA) and analyzed by mass spectrometry.
S.1.2.3. Mass Spectrometry: Samples were injected for separation by reverse phase chromatography on a NanoAcquity UPLC system (Waters, Dublin, CA). Peptides were loaded onto Symmetry ® C18 column (1.7 µm BEH-130, 0.1 x 100 mm, Waters, Dublin, CA) with a flow rate of 1 µL a minute and a gradient of 2% Solvent B to 25% Solvent B (where Solvent A is 0.1% Formic acid/2% ACN/water and Solvent B is 0.1% FA/2% water/ACN) applied over 60 min with a total analysis time of 90 min. Peptides were eluted directly into an Advance CaptiveSpray ionization source (Michrom BioResources/Bruker, Auburn, CA) with a spray voltage of 1.4 kV and analyzed in an LTQ Orbitrap mass spectrometer (ThermoFisher, San Jose, CA). Precursor ions were analyzed in the FTMS at 60,000 resolution. MS/MS was performed in the LTQ in data-dependent mode where the top 8 most abundant ions were subjected to fragmentation.
S.1.2.4. Bioinformatics: MS/MS data were searched using the Mascot Search Algorithm version 2.3.02 22. (Matrix Sciences, London, UK) against a concatenated forward-reverse target-decoy database
23
consisting of Homo sapiens proteins and common
contaminant sequences from UniProt (TrEMBL/SwissProt) release 2010_12.
assignments were first filtered to a 5% false discovery rate (FDR) using linear discriminant analysis and then to an overall 1% protein FDR using Protein Sieve15. Filtered data was quantified at the peptide level using the VistaQuant algorithm as previously described
23
. Relative abundance ratios at the protein
level were calculated from summed abundance measurements of peptides with VistaQuant Confidence Scores ≥ 9024. S.1.2.5 Informatic Processing a) SILAC Normalization and Quality Control AUC values from Vista were logarithmically transformed, then normalized to remove intensity-specific bias via an algorithm adapted to the SILAC context from the Yang et al. approach for normalizing cDNA microarray data
21
. For
peptides measured in each technical replicate, the paired heavy (H) and light (L) log-AUCs were transformed to their sum (H+L) and difference (H-L), where the difference is equivalent to the log-ratio between paired heavy and light AUCs. A so-called MA-scatterplot shows any systematic biases of log fold-change across intensity from that replicate. Such trends are estimated by a nonparametric smoother (in our case, the loess smoother) implemented in the R statistical language, R Development Core Team 201025 and subtracted out from the data before downstream analysis. A range of exploratory graphics including violin plots17 of the log-ratios and log-products, and graphs of numbers of peaks detected in each replicate by region were created to further understand the data and for informal quality control. All data can be located in Supplemental Tables S3(A-F).
b) Categorization of Differential Regulation
down-regulated) on those proteins with at least two peptides from each biological replicate. Proteins without two peptides per replicate were not analyzed via GASSP. Averaging was done via a linear mixed effects model (fit with the nlme package in R26, with separate random effects for peptides from distinct biological replicates. Based on its particular mixed effect model each protein was assigned two components of statistical variance: (1) an estimate of variance across biological replicates (this could be and sometimes was zero) and (2) an estimate of variance among peptides within a replicate (this was always nonzero). Across proteins, the nonzero estimates for each type of variance were shrunk towards their respective global geometric averages following a previously described approach23. Separately for each protein, the standard error attached to its estimated average log fold change was then
SEavg =
2 !! pept !! 2bio + # bio ! reps # peptides
2 2 where !! bio and !! pept are respectively the shrunken components of variance for
biological replicates and peptide variance derived from the Cui et al. global shrinkage procedure23. The differential regulations of proteins can be summarized from their average log fold changes and corresponding standard errors in a number of established ways (e.g., raw fold change, moderated t-statistic, confidence interval, p-value, q-value, etc.). A procedure we have found useful for categorizing proteins into (1) unchanged, (2) up-regulated, and (3) down-regulated groups is to first declare a (subjectively designated) zone of “ignorable” differential regulation (e.g., from 0.80 to 1.25, where a fold change of 1 -- a log fold change of 0 -- corresponds to
1.
Unchanged proteins were those for which the confidence interval for fold
change not only contains 1 (so the data are consistent with no differential expression), but also falls entirely within the designated zone of ignorable regulation (indicating that the true underlying differential expression is unlikely to be practically important). 2.
Up-regulated proteins were then those for which the confidence interval
falls entirely above the designated zone. 3.
Down-regulated proteins were those for which the confidence interval falls
entirely below the designated zone. c) Caspase Substrate Identification, Candidate caspase substrates were identified by associating SILAC log ratios for each peptide with gel migration information on that peptide via the following steps: 1. An approximate gel location G for each protein was identified based on the protein’s theoretical molecular weight and the observed gel locations of fifteen stock reference standard proteins with molecular weights ranging from 10 to 220 kDa. 2. It was observed that some proteins had unexpected peptide log ratios detected in gel bands corresponding to masses heavier (many times heavier, in some cases) than their theoretical masses. We speculate that these were due either to heavily glycosylated variants or mis-mappings. Influence of such unexpectedly heavy peptides was down-weighted by applying a one-sided Gaussian kernel filter to the data: if the i’th peptide for a protein with gel location G was observed in gel region xi , that peptide was weighted in the caspase substrate screening procedure by a factor of wi = exp(
− (G − xi ) + 2
)
The effect was to assign a weight of “1” to peptides found in gels predicted to contain peptides with mass at or below the protein’s theoretical mass, but to progressively down-weight the influence of data from gels containing peptides markedly heavier than expected from the full protein’s theoretical mass. 3. A robust linear model was fit, with the SILAC log ratio for peptides as the response and observed gel region (with previously defined weight wi for the i’th peptide) as the predictor. Robust linear model fits were obtained via the function rlm 27 from the MASS package version 7.3.14 in the R statistical language 22, with the weights described and other arguments to rlm set to their defaults. 4. Proteins selected for further follow-up were ones for which relatively strong evidence was present of a positive slope, i.e., that the log ratio of treated vs. untreated cells increased across gel positions containing peptides lighter than the protein’s nominal mass, consistent with treated cells having disproportionately more cleaved fragments in those gels. Arbitrary cut-offs for follow-up were based on t-statistics for the slope estimates from the robust linear model. In the HCT116 Bax screen, separate slopes were fit within the robust linear model for peptides measured in the KO and WT cells. Statistical procedures were used to rank proteins. Protein-specific plots of the raw peptide data across gels and biological replicates were then examined in order to prioritize selected proteins for follow up.
S.1.3 Immuno-Affinity enrichment of Caspase Substrate Peptides S.1.3.1 PTMscan Analysis of Caspase Substrates We performed an immuno-affinity isolation of caspase specific substrates using the PTMScan protocol16. PTMScan® Proteomics System was in-licensed
are derivatized with light and heavy reductive methylation16 to induce a quantitative label. Reductive methylation was performed during a solid phase extraction step on a C18 cartridge where tryptic digests of the untreated and etoposide treated Jurkat were reacted with CH2O(4%)/NaBH3CN(0.6 M) (Light) and CD2O/NaBD3CN (Heavy), respectively. The light and heavy samples were eluted with 40% ACN/0.1% TFA and combined prior to affinity purification using antibody recognizing the C-terminal aspartic acid residue. After lyophilization of eluted peptides, samples are resuspended in a previously described buffer and captured using antibodies that recognize the C-terminal aspartic acid motif. Resultant peptides are eluted in low pH and again purified using stage tips and resuspended in HPLC loading buffer & injected onto an Orbitrap mass spectrometer (please refer to section S.1.2.3 and S.1.2.4). S.1.3.1 Data analysis of PTMscan Analysis Subsets of peptides identified in the PTMscan experiment bearing the caspase substrate motif were displayed using Trestle. Trestle is an in-house developed, web-based application that allows the quantitative comparison of peptide or protein levels across various biological conditions. Both label-free and relative quantification such as SILAC are supported using this application. To remove sequence and charge state redundancy, peptides identified in an LC/MS run were mapped to extracted ion chromatographic peaks and only unique chromatographic peaks were summarized to quantitate peptide or protein abundance. The resulting quantitative measurements in each biological condition were visualized in a heat-map to highlight any substantial changes, along with additional supporting information to reveal the biological significance (data not shown). Data was exported from Trestle into Excel.
HCT 116 BAX -/- and HCT 116 BAX +/- were treated or untreated with 20 µM Z-FAD-FMK for 60 min followed by treatment with 2 µg/ml Apo2L for 120 min. Cells were harvested and lysed in 1% Triton-X-100 lysis buffer (50 mM Tris-HCl ph 8, 150 mM NaCl, 1% Triton-X-100), protein was quantified using a BCA assay (Pierce) according to manufacturer’s instructions. 30 µg of protein was run on 420% TGX gels (Bio-Rad) and then transferred to PVDF membrane. Membranes were blocked for 1hr in 5% milk in TBST and then probed with an antibody against BTF3 (Novus Biologicals), TFG (Novus Biologicals) or SQSTM1/p62 (Novus Biologicals). Blots were then probed with secondary antibodies for detection with either anti-mouse-HRP (Santa Cruz Biotech) or anti-rabbit-HRP (Jackson Labs.). Blots were stripped with Restore Plus Western Stripping Buffer (Pierce) according to manufacturer’s instructions and re-probed with an antibody against β-actin (Abcam) or α-tubulin (Cell Signaling Tech.) and then visualized with secondary anti-rabbit-HRP antibody for loading controls. Supplemental Figures S1: A representation of the gel bands excised in a typical GASSP experiment S2. Full list of proteins characterized using GASSP categorized as having a) no change in expression level b) increase fold change c) a decreased fold change and d) proteolysis after treatment of Jurkat cells with etoposide. S3. BAAT1 was identified as a caspase substrate using the PTMscan approach. This protein would not have been identified using the GASSP approach as only a small truncation of the C-terminus was observed after proteolysis, Conversely, PARP1 was identified using the GASSP approach but not identified using the PTMscan approach. This was because the tryptic peptide at the caspase
S4: Proteolysis substrates identified using GASSP from PARA induced apoptosis in BAX (-/+) and BAX (-/-) HCT116 isogenic cell lines. S5: Venn diagram demonstrating the overlap between all the substrates identified from the PARA induced apoptosis in BAX (-/+) and (-/-) HCT116 cell lines (pink) versus previous proteomic studies (green)11, 12. S6: Venn diagram showing the overlap between the substrates identified from the PARA induced apoptosis in BAX (-/+) and (-/-) HCT116 cell lines (pink) versus the CASBAH (green)18. S7: Western blot data confirming that the proteins (a) BTF3, (b) PARP1, (c) caspase 2 are post-mitochondrial (BAX -/+) caspase-mediated proteolytic substrates.
Table S1 (a-d) GASSP data for etoposide induced apoptosis in Jurkat cells. Table S2: HCT116 PARA induced apoptotic substrates using the PTMscan approach Tables S3: located at http://research-pub.gene.com/jlill_data/ (a)
GASSP BAX (-/-) heavy SILAC labeled + APO2L treatment, light SILAC labeled untreated, (b), GASSP BAX (-/-) light SILAC labeled + APO2L treatment, heavy SILAC labeled untreated,(c) GASSP BAX (+/-) heavy SILAC labeled + APO2L treatment, light SILAC labeled untreated,(d) GASSP BAX (+/-) light SILAC labeled + APO2L treatment, heavy SILAC labeled untreated, (e) GASSP Jurkat light SILAC labeled etoposide treated, heavy SILAC untreated, (f) GASSP Jurkat heavy SILAC labeled
