Supplemental table 1. The completeness of trypsin digestion of the signature peptides with native flanking sequence. The signature peptides with native flanking sequence were digested with trypsin as described in the Experimental Section. The amount of each peptide before and after trypsin digestion was analyzed by SRM-MS. The trypsin digestion completeness was calculated as the remaining percentage of the peptides after trypsin digestion. ND: not detectable. Protein Sequence Before trypsin digestion After digestion with trypsin for 24 h digestion a Peak intensity Trypsin Mean S.D. CV% Mean S.D. CV% completeness MAVS LTK-VSASTVPTDGSSR-NEE 1.84E+8 252384.1.1% 1734 5444.722 5.1% 99.94% RIG-I KGK-VVFFANQIPVYEQQK-SVFS 781284 49879.31 6.4% ND b - - 1.% IKKA DSK-IQLPIIQLRK-VW 95177359 16927 1.8% 29552.5 687.482 2.6% 99.97% NIK CLK-IASEPPPVR-EIP 9449289 599432.1 6.3% 3633 565.6854 15.6% 99.96% p52/p1 PSR-DSGEEAAEPSAPSR-TPQ 67818182 1544634 2.3% 243898.5 5189.457 2.1% 99.64% p1 SAR-ALLDYGVTADAR-ALL 43114176 1449659 3.4% 148691 391.64 2.2% 99.66% IKKB FFK-TSIQIDLEK-YSE 691759.5 4847.44 5.9% 22 33.94113 1.7% 99.71% IKKE TER-FSSVAGTPEIQELK-AAA 16638 5114.7 3.1% ND c - - 1.% IRF3 GLR-LVGSEVGDR-TLP 4129166 262283.6.6% 29752.5 3246.327 1.9% 99.93% TBK1 GLR-LVGSEVGDR-TLP 137425 284291.6 2.2% ND c - - 1.% RelA VFR-TPPYADPSLQAPVR-VSM 2.84E+9 391217 1.4% 668589 132852.6 2.% 99.77% IKBa QEIR-LEPQEVPR-GSE 9896976 141313 14.3% 1142.245 218.8566 19.2% 99.99% a. The peptide sequences in bold are signature peptides. b. Noise level is about 1 counts. c. Noise level is about 2 counts.
Supplemental Table 2. Activation of noncanonical NF-κB pathway by the stimulation of ds-rna. A549 cells were treated with ds-rna for, 1 and 4 h, or treated with TNF for,.5 and 1 h, respectively. The nuclear proteins were extracted and subjected to SID-SRM-MS analysis. Each sample was analyzed by LC-SRM-MS twice. All data were manually inspected to ensure peak detection and accurate integration as described in Experimental Procedure section. The peak area of extract ion chromatogram of native peptides and SIS-peptides of p52 and p1 were integrated using Xcalibur 2.1. The default values for noise percentage and base-line subtraction window were used. The ratio between the peak area of native and SIS version of each peptide were calculated. Beta-actin was used as loading control. All of the measured native peptide versus SIS-peptide ratios were normalized by beta-actin. The native verse SIS-peptide ratios measured in the two replicate LC-SRM-MS analysis were averaged, and a mean and standard deviation (SD) were calculated. Stimuli ds-rna TNF p52 p1 Time (h) Mean ratio Mean ratio SD (native/sis-peptide) (native/sis-peptide) SD.384.79.38.6 1.45.31.35.2 4.67.64.34.11.656.77.32.4.5.47.12.27.8 1.31.23.23.4
Supplemental Figure 1. Overview of innate immune response highlighting the NF-κB and IRF3 pathways. Shown is a schematic view of the IIR displaying the canonical NF-κB, noncanonical NF-κB and IRF3 signaling pathways of the IIR. The proteins listed are targeted for SID-SRM assay development. Abbreviations: IKK, IκB kinase, IκBα, inhibitor of IκBα isoform; RIG-I, retinoic acid inducible gene-i; MAVS, mitochondrial antivirals signaling; TBK1, Tank binding kinase 1.
Supplemental Figure 2. Evaluation of the specificity of SRM assays. The SIS peptides were spiked in the trypsin digests of nuclear fraction. The native and SIS peptide were analyzed by SRM. In each figure, the left panels are the extract ion chromatogram, and the right panels are the SRM spectra.
RT: 19.6 628.38 16 ACTB-Native 12 ACTB-Native 14 1 12 8 1 8 6 6 4 531.32 775.45 4 2 2 RT: 19.6 638.38 874.51 55 5 ACTB-SIS 4 ACTB-SIS 45 35 4 3 35 3 25 25 2 2 15 1 5 14 16 18 2 22 24 15 541.32 785.45 1 5 884.51 5 6 7 8 9
RT: 17.1 867.5 RelA-Native 1 RelA-Native 14 9 12 8 1 7 8 6 5 6 4 4 3 1313.68 2 2 1 982.53 153.57 1 9 RT: 16.97 RelA-SIS 6 877.5 RelA-SIS 8 5 7 6 5 4 3 4 3 2 1323.68 2 1 1 992.53 163.57 1 15 2 25 3 8 1 12 14
4 35 3 25 2 15 RT: 1.98 IRF3-Native 3 25 2 15 IRF3-Native 719.33 1 5 3 25 8.67 1.9 12.29 14.29 1.32 11.29 13.36 RT: 1.98 IRF3-SIS 14.37 1 5 25 446.23 IRF3-SIS 575.28 662.31 729.33 2 15 2 15 1 12.63 1 5 8.78 9.48 1.29 11.25 13.44 14.6 8 9 1 11 12 13 14 15 5 456.23 456.23 585.28 672.31 585.28 672.31 729.33
5 45 4 35 3 25 2 14.14 14.86 19.29 p1-native 21.61 45 4 35 3 25 2 689.36 852.42 967.45 p1-native 15 15 1 5 RT: 18.51 11.71 16.4 22.88 24.5 26.4 1 5 18.53 16 14 12 RT: 18.48 p1-sis 6 5 699.36 862.42 977.45 p1-sis 1 8 4 3 19.53 6 4 1 2 14.69 2.2 21.35 26.1 11.83 29.72 1 15 2 25 3 2 7 8 9 1 11
5 p1/p52-native 17.1 7 614.33 p1/p52-native 6 4 17.68 5 3 2 1 13.91 RT: 1.99 4 3 2 1 359.2 743.37 814.41 885.44 114.48 1.87 4.25 9.54 9.95 RT: 1.96 624.33 2 p1/p52-sis 18 16 p1/p52-sis 15 1 14 12 1 8 6 824.41 5 18.63 15.19 3.21 4.71 1.9 5 1 15 2 4 2 369.2 753.37 895.44 124.48
11.75 725.39 IκBα-Native RT: 12.18 14 IκBα-Native 2 12 15 1 1 8 6 5 1.67 11.17 12.6 13.21 14.68 4 2 5.28 628.34 8.33 9.56 1.33 RT: 12.14 735.39 854.44 967.52 12 IκBα-SIS 7 IκBα-SIS 1 6 8 6 5 4 4 1.71 3 2 51.28 638.34 2 1 11.14 12.33 13.14 8.71 9.21 1.14 8 9 1 11 12 13 14 15 864.44 977.52 6 8 1
Supplemental Figure 3. Evaluation of the specificity of SRM assays. The SIS peptides were spiked in the trypsin digests of cytoplasma fraction. The native and SIS peptide were analyzed by SRM. In each figure, the left panels are the extract ion chromatogram, and the right panels are the SRM spectra.
RT: 18.6 628.38 6 ACTB-Native 35 ACTB-Native 5 3 4 25 3 2 2 15 531.32 775.45 1 1 5 3 RT: 18.64 ACTB-SIS 18 638.38 ACTB-SIS 874.51 25 16 14 2 15 12 1 8 541.32 1 6 785.45 5 RT: 12.91 14 16 18 2 22 24 4 2 884.51 5 6 7 8 9
45 4 IκBα-Native RT: 1.56 24 22 2 IκBα-Native 725.39 35 18 3 16 5.28 25 2 15 1.6 14 12 1 8 628.34 1 5 1.51 2.55 6.82 12.18 9.6 RT: 1.52 16.37 19.26 6 4 2 735.39 854.44 967.52 11 1 IκBα-SIS 6 IκBα-SIS 9 8 5 7 6 5 4 3 51.28 4 3 15.1 2 1 9.83 14.3 16.14 1.51 4.9 9.29 5 1 15 2 2 1 638.34 864.44 977.52 6 8 1
18 16 14 12 1 8 15.42 17.61 RT: 23.5 IKKα-Native 14 12 1 8 6 739.48 IKKα-Native 852.57 6 4 4 2 35 18.42 24.31 22. 29.78 26. 28.97 RT: 23.5 IKKα-SIS 2 2 529.34 642.43 749.48 IKKα-SIS 3 18 25 2 15 16 14 12 1 8 862.57 1 6 5 4 2 539.34 652.43 15 2 25 3 5 6 7 8 9
12 IRF3-Native 12.13 IRF3-Native 719.33 1 6 8 RT: 11.9 5 6 4 3 4 2 12.28 4.16 IRF3-SIS 9.21 1.94 RT: 11.13 15.37 17.72 2 1 1 446.23 IRF3-SIS 575.28 662.31 729.33 1 8 8 6 4 6 4 2 5 1 15 2 2 456.23 456.23 585.28 585.28 672.31 672.31 729.33
RT: 1.76 719.33 9 MAVS-Native 7 MAVS-Native 8 6 7 5 6 5 4 4 3 3 2 2 1 1 818.4 919.45 16.48 RT: 1.77 729.33 7 MAVS-SIS 55 5 MAVS-SIS 6 45 5 4 35 4 3 3 25 2 2 15 1 1 5 828.4 929.45 116.48 8 1 12 14 16 7 8 9 1 11
RT: 21.37 133.49 18 Nemo-Native 1 Nemo-Native 16 9 14 8 12 7 1 8 6 6 5 4 3 1146.57 4 2 2 1 976.47 1259.66 1346.69 3 RT: 21.33 Nemo-SIS 18 16 143.49 Nemo-SIS 25 14 2 12 15 1 8 1156.57 1 6 1356.69 5 4 2 986.47 1269.66 1 15 2 25 3 1 11 12 13 14
8 14.32 p1-native 7 689.36 p1-native 7 6 967.45 6 5 4 3 2 14.81 RT: 18.71 21.73 5 4 3 2 852.42 18.53 1 11.39 25.78 17.79 29.4 1 1 9 8 RT: 18.69 p1-sis 4 35 3 699.36 862.42 977.45 p1-sis 7 6 5 25 2 19.53 4 15 3 1 2 1 5 1 15 2 25 3 7 8 9 1 11
2 p52/p1-native 17.3 8 614.33 7 15 6 1 RT: 11.25 5 4 3 814.41 5.92 2.63 2 8.73 1.67 RT: 11.28 16.52 18.84 2 1 359.2 624.33 743.37 885.44 114.48 18 14 16 12 14 12 1 8 6 4 2 15.1 17.74.92 5.78 1.27 5 1 15 2 1 8 6 4 2 369.2 753.37 824.41 895.44 124.48
2 18 RT: 16.36 RelA-Native 12 867.5 RelA-Native 16 1 14 12 1 8 6 8 6 4 1313.68 4 2 2 982.53 153.57 RT: 16.36 877.5 6 RelA-SIS 35 RelA-SIS 5 3 4 3 2 25 2 15 1 1323.68 1 15.9 23.13 11.85 16.74 18.52 2.52 1 12 14 16 18 2 22 24 992.53 163.57 5 8 1 12 14
25 RT: 21.87 RIG-I-Native 14 891.46 RIG-I-Native 2 12 1 15 1 8 6 5 4 2 14.54 RT: 21.99 91.46 1132.6 1246.64 35 RIG-I-SIS 14 RIG-I-SIS 3 12 25 1 2 15 8 6 1 4 5 2.1 2.29 23.7 16.33 23.8 29.26 29.61 15 2 25 3 35 2 114.54 1142.6 1256.64 9 1 11 12 13
14 2.32 2.78 TBK1-Native 35 3 817.49 TBK1-Native 12 25 1 8 6 RT: 17.85 28.9 2 15 4 2 45 4 12.69 23.44 13.89 26.71 16.8 25.78 RT: 17.82 TBK1-SIS 1 5 25 827.49 931.53 16.58 1189.62 TBK1-SIS 35 2 3 25 2 15 25.9 15 1 941.53 1 5 19.1 16.58 13.8 22.43 27.59 1 15 2 25 3 1199.62 5 17.58 8 9 1 11 12
Supplemental Figure 4. SDS-PAGE analysis of recombinant IIR target proteins before and after trypsin digestion. The mixture of recombinant IIR target proteins was digested with trypsin for 24 h. The recombinant protein mix and tryptic digest were loaded on the 1% SDS-PAGE gel. After electrophoresis, the gel was stained with SYPRO Ruby staining (A). Line 2 is the recombinant protein mix before trypsin digestion; Line 4 is the tryptic digest of the recombinant protein mix. Line 1 and 3 are Precision Plus Protein Duel Color standard. Because the MW maker was prestained, some MW maker protein bands cannot be visualized by SYPRO Ruby. The same gel was later stained with Colloidal Bule (Invitrogen) to demonstrate the protein bands of protein MW markers (B).
Supplemental Figure 5. Time course analysis of the release of signature peptides during tryptic digestion. A mixture of recombinant IIR target proteins was prepared for the time-course digestion. The proteins were denatured with 8 M Guanidine HCl, and reduced and alkylated as described in the Experimental Section. Before trypsin digestion, a mixture of SIS peptides of the signature peptides of IRF3, IkBa, IKKr, RelA, MAVS, IKKa, TBK1, RIG-I, and p1/p52 was added in the mixture. The sample was diluted 1 times with 5 mm ammonium bicarbonate. Trypsin was added in the sample followed by incubation incubation at 37 C. At s, 5 min, 1 min, 3 min, 45 min, 1 h, 2 h, 3 h, 4 h, 5 h, 8 h, 12 h, 16 h, 2 h, 24 h, and 3 h, 1 µl portion of tryptic digest was removed and mixed with 2 µl of trifluoroacetic acid and refrigerated to terminate proteolysis. Each portion was analyzed by SID-SRM-MS twice. The native vs SIS-peptide ratio of each signature peptide was calculated. The error bar is the standard deviation of the two replicates.
Supplemental Figure 6. Schematic representation of the workflow for analysis of IIR in response to the stimulation of ds-rna. A549 cells are treated with ds-rna for varying times. The cells were fractionated into cytoplasmic and nuclear fractions. The proteins were extracted and digested with trypsin. The SIS peptides of target proteins were spiked into each tryptic digest. The peptide mixtures were analyzed by LC-SRM-MS. Finally, the temporal profile of each target protein was generated based on the SID-SRM-MS analysis.
Supplemental Figure 7. SRM analysis of Lamin B in cytoplasmic fraction and nuclear fraction. 1
Supplemental Figure 8. Cross talk of NFkB and IRF3 pathways. A549 cells were transfected with dsrna in the absence of specific sirna (Con sirna), or sirna targeting RelA or IRF3 as indicated. A. Effect of RelA and IRF3 knockdown on RelA expression. Note that IRF3 knockdown upregulates RelA expression. B. Effect of RelA and IRF3 knockdown on RelA and IRF3 expression. Note upregulation of RelA with IRF3 knockdown. C. Effect of RelA and IRF3 knockdown on ISG56 expression. ISG56 is almost completely IRF3 dependent. Note upregulation of ISG56 expression with RelA knockdown. D. Effect of RelA and IRF3 knockdown on IL8 expression. IL8 is almost completely RelA dependent. Note upregulation of IL8 with IRF3 knockdown. A B C D