Dataset DOI: 10.5061/dryad.8w9ghx43t

Description of the data and file structure

This dataset contains the raw mass spectrometry files underlying the ultra‑performance liquid chromatography coupled with high‑resolution mass spectrometry (UPLC‑HRMS) and tandem mass spectrometry (MS/MS) analyses reported in the associated research article. All mass-spec data are provided in the mzML format, which can be imported into certain free/open software (such as the "Skyline", https://skyline.ms/project/home/software/Skyline/begin.view), for extracted ion chromatogram (EIC) extraction, peak-area integration, and inspection of MS1 and MS/MS spectra.

Files and variables

File: Fig.2A.zip

Description: 

The data were acquired by ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC–HRMS) to assess depletion of oxidized nicotinamide adenine dinucleotide (NAD⁺). The experimental conditions include the wild-type (WT) Toll/interleukin-1 receptor (TIR) domain of Toll-like receptor 4 (TLR4), its E748Q mutant, the positive controls AbTIR and SARM1, and a negative control. E748Q denotes substitution of glutamate with glutamine at residue 748.

The archive contains the following mzML files: abtir+nad-1.mzML, abtir+nad-2.mzML, abtir+nad-3.mzML, Ctrl-nad-1.mzML, Ctrl-nad-2.mzML, Ctrl-nad-3.mzML, Sarm1+NAD-1.mzML, Sarm1+NAD-2.mzML, Sarm1+NAD-3.mzML, TLR4-TIR(E748Q)+nad-1.mzML, TLR4-TIR(E748Q)+nad-2.mzML, TLR4-TIR(E748Q)+nad-3.mzML, TLR4-TIR(WT)+nad-1.mzML, TLR4-TIR(WT)+nad-2.mzML, and TLR4-TIR(WT)+nad-3.mzML. The numbers 1–3 indicate three biological replicates for each experimental condition. “Ctrl-nad” identifies the negative control; “abtir+nad” and “Sarm1+NAD” represent the positive controls containing AbTIR, a TIR domain protein from Acinetobacter baumannii, and SARM1, sterile alpha and TIR motif-containing protein 1, respectively.

File: Fig.2E_and_2F.zip

Description: 

This compressed archive contains UPLC–HRMS full-scan mass spectrometry (MS1) data in mzML format for the analysis of small molecules bound to TLR4-TIR proteins immobilized on agarose beads. The archive contains the following files: ADPR (Std).mzML, Ctrl.mzML, TLR4-TIR(E748Q).mzML, and TLR4-TIR(WT).mzML.

ADPR (Std).mzML contains the MS1 data acquired from the ADP-ribose (ADPR) standard, whereas the remaining files contain the MS1 data acquired from the indicated experimental samples.

The associated figures show MS1 spectra extracted over a retention time window of 2.1–2.3 minutes (E) and targeted extracted ion chromatograms (EICs) (F). Compared with the negative control, TLR4-TIR(WT) showed a distinct ion peak at m/z 558.0664.

File: Fig.2G.zip

Description: 

This archive contains two files: ADPR.mzML and TLR4.mzML.

ADPR.mzML contains the tandem mass spectrometry (MS/MS) data acquired from the ADP-ribose (ADPR) standard, whereas TLR4.mzML contains the MS/MS data acquired from the TLR4-TIR sample.

The MS/MS spectra shown in panel G were obtained by collision-induced dissociation (CID) of the ion at m/z 558.0644 (z = −1), with the ADPR standard used as the control. The TLR4-TIR sample showed a fragmentation pattern matching that of the ADPR standard, identifying the detected product as ADPR.

File: Fig.3J.zip

Description: 

This archive contains three files: ADPR (Std).mzML, Peak A.mzML, and Peak B.mzML.

ADPR (Std).mzML contains the UPLC–HRMS full-scan mass spectrometry (MS1) data acquired from the ADP-ribose (ADPR) standard. Peak A.mzML and Peak B.mzML contain the MS1 data acquired from the size-exclusion chromatography (SEC) fractions designated as Peak A and Peak B, respectively, in Fig. 3G and Fig. 3H of the associated research article.

Targeted extracted ion chromatogram (EIC) analysis of these SEC fractions (Fig. 3J) showed that ADPR was associated with the higher-order TLR4–TIRAP–MyD88 TIR complex but not with monomeric TIRAP-TIR.

File: Fig.4I.zip

Description: 

These data were acquired to analyze reaction products from bead-immobilized TLR4-TIR proteins, including the wild-type protein and the indicated mutants (C745Y, E748Q, H706W, S742A, and W744D), incubated with NAD⁺.

This archive contains the following files: C745Y+NAD_1.mzML to C745Y+NAD_3.mzML, CTRL+NAD_1.mzML to CTRL+NAD_3.mzML, E748Q+NAD_1.mzML to E748Q+NAD_3.mzML, H706W+NAD_1.mzML to H706W+NAD_3.mzML, S742A+NAD_1.mzML to S742A+NAD_3.mzML, TLR4+NAD_1.mzML to TLR4+NAD_3.mzML, and W744D+NAD_1.mzML to W744D+NAD_3.mzML.

“CTRL+NAD” represents the control, and “TLR4+NAD” represents the wild-type TLR4-TIR sample. The suffixes _1, _2, and _3 distinguish the three replicate samples for each experimental condition.

The relative abundance of the ADP-ribose (ADPR) product was quantified using the extracted ion chromatogram (EIC) at m/z 558.0644 (z = −1). Mutations of key TLR4 residues involved in ADPR binding significantly reduced or abolished NADase activity.

File: Fig.5F_and_5G.zip

Description: 

These data were acquired to analyze reaction products from bead-immobilized TLR4-TIR proteins, including the wild-type protein and the indicated mutants targeting the BCD interface, incubated with NAD⁺.

This archive contains the following files: CTRL+NAD_1.mzML to CTRL+NAD_3.mzML, E748Q+NAD_1.mzML to E748Q+NAD_3.mzML, F747D+NAD_1.mzML to F747D+NAD_3.mzML, I721D+NAD_1.mzML to I721D+NAD_3.mzML, I751D+NAD_1.mzML to I751D+NAD_3.mzML, and TLR4+NAD_1.mzML to TLR4+NAD_3.mzML.

“CTRL+NAD” represents the control, and “TLR4+NAD” represents the wild-type TLR4-TIR sample. The suffixes _1, _2, and _3 distinguish the three replicate samples for each experimental condition.

Panel F shows the extracted ion chromatograms (EICs) at m/z 558.0644 (z = −1). The data shown are representative of three independent experiments. Panel G shows the quantification of relative ADPR abundance. Mutations at the TLR4 dimerization interface significantly reduced NADase activity, indicating that TLR4 NADase activity depends on dimerization.

File: Fig.6C.zip

Description: 

The archive contains the following files: ADPR std.mzML, myd88-1.mzML to myd88-3.mzML, R808D-1.mzML to R808D-3.mzML, R808D-TIRAP-Myd88-1.mzML to R808D-TIRAP-Myd88-3.mzML, TIRAP-1.mzML to TIRAP-3.mzML, TLR4-TIRAP-Myd88-1.mzML to TLR4-TIRAP-Myd88-3.mzML, TLR4-WT-1.mzML to TLR4-WT-3.mzML, Y792D-1.mzML to Y792D-3.mzML, and Y792D-TIRAP-Myd88-1.mzML to Y792D-TIRAP-Myd88-3.mzML.

ADPR std.mzML contains the MS1 data acquired from the ADPR standard. Files named “TLR4-TIRAP-Myd88”, “R808D-TIRAP-Myd88”, and “Y792D-TIRAP-Myd88” contain data from the ternary complexes containing WT TLR4, TLR4(R808D), and TLR4(Y792D), respectively. The suffixes -1, -2, and -3 indicate three biological replicates for each experimental condition.

Endogenous ADPR associated with the purified proteins shown in Fig. 6B was quantified by mass spectrometry. Relative ADPR abundance was normalized to TLR4 protein levels, as determined by densitometric analysis of the Coomassie-stained gel in Fig. 6B using ImageJ.

ADPR was detected in the mutant-containing ternary complexes, despite the inability of the mutants alone to produce ADPR. These results strongly suggest that ADPR production during complex assembly depends on the heterotypic TLR4–TIRAP interface.

File: Fig.6D_and_6E.zip

Description: 

These data were acquired to analyze reaction products from bead-immobilized TLR4-TIR proteins, including the wild-type (WT) protein and the E748Q, R808D, and Y792D mutants, incubated with NAD⁺.

The archive contains the following files: ADPR std.mzML, E748Q+NAD_1.mzML to E748Q+NAD_3.mzML, R808D+NAD_1.mzML to R808D+NAD_3.mzML, TLR4+NAD_1.mzML to TLR4+NAD_3.mzML, and Y792D+NAD_1.mzML to Y792D+NAD_3.mzML.

ADPR std.mzML contains the MS1 data acquired from the ADP-ribose (ADPR) standard. “TLR4+NAD” represents the wild-type TLR4-TIR sample. The suffixes _1, _2, and _3 distinguish the three replicate samples for each experimental condition.

Panel D shows the extracted ion chromatograms (EICs) at m/z 558.0644 (z = −1), with the ADPR standard used as a reference. Panel E shows the quantification of relative ADPR abundance. The E748Q, R808D, and Y792D mutants showed reduced NADase activity compared with wild-type TLR4.

File: Fig.6F_and_6G.zip

Description: 

This archive contains the following files: ADPR.mzML, R808D-1.mzML to R808D-3.mzML, and R808D-TIRAP-Myd88-1.mzML to R808D-TIRAP-Myd88-3.mzML.

R808D-1.mzML to R808D-3.mzML contain the MS1 data acquired from the TLR4(R808D) samples, whereas R808D-TIRAP-Myd88-1.mzML to R808D-TIRAP-Myd88-3.mzML contain the MS1 data acquired from the corresponding ternary complex samples. The suffixes -1, -2, and -3 distinguish the three replicate samples for each experimental condition.

Individually purified GST-TLR4-TIR(R808D), Strep-tag II-TIRAP-TIR, and 6×His-SUMO-MyD88-TIR proteins were mixed with NAD⁺. The reassembled ternary complex was then isolated by Strep-Tactin affinity purification and analyzed by mass spectrometry. Bead-immobilized GST-TLR4-TIR(R808D) protein was used as the control.

Panel F shows the extracted ion chromatograms (EICs) at m/z 558.0644 (z = −1), and panel G shows the quantification of relative ADPR abundance. Adding TIRAP-TIR and MyD88-TIR in vitro restored the NADase activity of the TLR4-TIR(R808D) mutant. These results strongly suggest that ADPR production during complex assembly depends on the heterotypic TLR4–TIRAP interface.

File: Fig.S5B_and_S5C.zip

Description: 

These data were acquired to analyze small molecules bound to bead-immobilized wild-type (WT) TLR4-TIR and the catalytically inactive TLR4-TIR(E748Q) mutant after incubation with NAD⁺.

The archive contains the following files: E748Q+NAD_1.mzML to E748Q+NAD_3.mzML and TLR4+NAD_1.mzML to TLR4+NAD_3.mzML.

“E748Q+NAD” represents the TLR4-TIR(E748Q) samples, and “TLR4+NAD” represents the wild-type TLR4-TIR samples. The suffixes _1, _2, and _3 distinguish the three replicate samples for each experimental condition.

Panel B shows the HRMS profiles over the retention time window of 2.0–2.3 minutes for three replicates per group. Panel C shows a volcano plot comparing m/z features between the WT and E748Q groups based on the data in panel B. Raw MS data were preprocessed by rounding m/z values to three decimal places, and features within ±0.005 Da were computationally binned as identical ions. Each feature’s fold change (WT/E748Q MS intensity) is plotted against its adjusted p-value. HRMS analysis of this LC peak revealed a singly charged ion at m/z 558.0664 (z = −1).

File: Fig.S5D_left.zip

Description: 

This archive contains data for the flow-through (FT), protein-free supernatant samples from the control, TLR4, TLR4(E748Q), and SARM1 groups analyzed with or without NAD⁺.

The archive contains the following files: ctr_ft_1.mzML to ctr_ft_3.mzML, ctrl+NAD_ft_1.mzML and ctrl+NAD_ft_2.mzML, e748q_ft_1.mzML to e748q_ft_3.mzML, e748q+nad_ft_1.mzML and e748q+nad_ft_2.mzML, sarm1_ft_1.mzML to sarm1_ft_3.mzML, sarm1+nad_ft_1.mzML to sarm1+nad_ft_3.mzML, tlr4_ft_1.mzML to tlr4_ft_3.mzML, and tlr4+nad_ft_1.mzML to tlr4+nad_ft_3.mzML. The numerical suffixes distinguish replicate samples within each experimental condition.

The associated experiment quantified ADP-ribose (ADPR) in protein-free supernatants after the reaction. In-bead enzymatic assays were performed with or without the NAD⁺ substrate. The protein-free supernatants were analyzed separately by LC–HRMS, and the corresponding MS1 data are included in this archive. An elevated baseline of ADPR in the supernatant samples indicates spontaneous NAD⁺ degradation during the reactions.

File: Fig.S5D_right.zip

Description: 

These data were acquired to analyze bead-associated reaction products from the control, TLR4, TLR4(E748Q), and SARM1 samples in the presence or absence of NAD⁺.

The archive contains the following files: CTR-1.mzML to CTR-3.mzML, CTRL+NAD_1.mzML to CTRL+NAD_3.mzML, E748Q-1.mzML to E748Q-3.mzML, E748Q+NAD_1.mzML to E748Q+NAD_3.mzML, Sarm1-1.mzML to Sarm1-3.mzML, Sarm1+NAD_1.mzML to Sarm1+NAD_3.mzML, TLR4-1.mzML to TLR4-3.mzML, and TLR4+NAD_1.mzML to TLR4+NAD_3.mzML. The numerical suffixes 1–3 distinguish the three replicate samples for each experimental condition.

The associated experiment quantified ADP-ribose (ADPR) in protein-bound bead fractions after the reaction. In-bead enzymatic assays were performed in triplicate with or without the NAD⁺ substrate. The glutathione agarose beads carrying the proteins from each reaction were analyzed separately by LC–HRMS, and the corresponding MS1 data are included in this archive. Pre-bound ADPR was also detected on beads carrying wild-type TLR4-TIR, suggesting that it may have originated from intracellular NAD⁺ hydrolysis during TLR4-TIR overexpression in insect cells.

File: Fig.S5E_and_S5F.zip

Description: 

These data were acquired to analyze nicotinamide (NAM) in protein-free supernatants after the reaction. The samples include the control, TLR4(E748Q), and wild-type (WT) TLR4 flow-through fractions, together with a NAM standard.

The archive contains the following files: CTRL-F-1.mzML to CTRL-F-3.mzML, EQ-F-1.mzML to EQ-F-3.mzML, t4-F-1.mzML to t4-F-3.mzML, and NAM.mzML.

NAM.mzML contains the MS1 data acquired from the nicotinamide standard. “CTRL” represents the buffer control, “EQ” represents the TLR4(E748Q) mutant, “t4” represents wild-type TLR4, and “F” represents the flow-through fraction. The numerical suffixes -1, -2, and -3 distinguish the three replicate samples for each experimental condition.

Panel E shows targeted extracted ion chromatograms (EICs) of the ion at m/z 123.0553 (z = +1) in the protein-free supernatants, acquired in positive electrospray ionization (ESI) mode, with the NAM standard used as a reference. Panel F shows the quantification of NAM production in the protein-free supernatants. Consistent with NAD⁺ hydrolysis, wild-type TLR4-TIR produced higher levels of NAM in the supernatant than the buffer control or the E748Q mutant.

File: Fig.S6C_S6D_andS6G_right.zip

Description: 

The experiments used the NAD⁺ derivative nicotinamide-8-bromoadenine dinucleotide (8-Br-NAD⁺) as the substrate to examine its hydrolysis by bead-immobilized TLR4-TIR. The samples include 8-Br-ADPR and 8-Br-NAD⁺ standards, controls, wild-type (WT) TLR4, the TLR4(E748Q) mutant, and SARM1, with the indicated protein samples analyzed in the presence or absence of 8-Br-NAD⁺.

The archive contains the following files: Br-ADPR_standard.mzML, Br-NAD-standard.mzML, CTR-1.mzML to CTR-3.mzML, ctrl+Br-NAD_1.mzML to ctrl+Br-NAD_3.mzML, E748Q+Br-NAD_1.mzML to E748Q+Br-NAD_3.mzML, E748Q-1.mzML to E748Q-3.mzML, Sarm1+Br-NAD_1.mzML to Sarm1+Br-NAD_3.mzML, Sarm1-1.mzML to Sarm1-3.mzML, Tlr4+Br-NAD_1.mzML to Tlr4+Br-NAD_3.mzML, and TLR4-1.mzML to TLR4-3.mzML.

Br-ADPR_standard.mzML and Br-NAD-standard.mzML contain the MS1 data acquired from the 8-Br-ADPR and 8-Br-NAD⁺ standards, respectively. The numerical suffixes 1–3 distinguish the three replicate samples for each experimental condition.

Panel C shows the LC–HRMS profiles over a retention time window of 3.3–3.6 minutes, and panel D shows targeted extracted ion chromatograms (EICs). Panel G shows the quantification of 8-Br-ADPR production in the protein-bound bead fractions after the reaction.

The product generated by TLR4-TIR matched the expected 8-Br-ADPR compound in retention time and molecular mass. The 8-Br-ADPR product was exclusively associated with TLR4-TIR on the beads, whereas the SARM1-TIR control generated 8-Br-ADPR in the supernatant with higher efficiency.

File: Fig.S6E.zip

Description: 

These data were acquired to compare the fragmentation pattern of the product generated by TLR4-TIR in the presence of 8-Br-NAD⁺ with that of the 8-Br-ADPR standard.

The archive contains Br-ADPR_standard_MSMS.mzML, which contains the MS/MS data acquired from the 8-Br-ADPR standard, and Tlr4+Br-NAD_MSMS_1.mzML to Tlr4+Br-NAD_MSMS_3.mzML, which contain the MS/MS data acquired from the TLR4 samples incubated with 8-Br-NAD⁺. The numerical suffixes _1, _2, and _3 distinguish the three replicate samples for this experimental condition.

The MS/MS spectra were obtained by collision-induced dissociation (CID) of the ion at m/z 635.9749 (z = −1). The product generated by TLR4-TIR showed an MS/MS fragmentation pattern matching that of the 8-Br-ADPR standard.

File: Fig.S6G_left.zip

Description: 

This archive contains the following files: Br-ADPR_standard.mzML, Br-NAD-standard.mzML, ctrl_ft_1.mzML to ctrl_ft_3.mzML, ctrl+br-nad_ft_1.mzML to ctrl+br-nad_ft_3.mzML, e748q_ft_1.mzML to e748q_ft_3.mzML, e748q+br-nad_ft_1.mzML to e748q+br-nad_ft_3.mzML, sarm1_ft_1.mzML to sarm1_ft_3.mzML, sarm1+br-nad_ft_1.mzML to sarm1+br-nad_ft_3.mzML, tlr4_ft_1.mzML to tlr4_ft_3.mzML, and tlr4+br-nad_ft_1.mzML to tlr4+br-nad_ft_3.mzML.

Br-ADPR_standard.mzML and Br-NAD-standard.mzML contain the MS1 data acquired from the 8-Br-ADPR and 8-Br-NAD⁺ standards, respectively. “FT” represents the flow-through fraction, and the numerical suffixes _1, _2, and _3 distinguish the three replicate samples for each experimental condition.

The associated experiment quantified 8-Br-ADPR production in protein-free supernatants after the reaction. This archive contains the MS1 data for the protein-free supernatant (FT) samples and the standards. The 8-Br-ADPR product was exclusively associated with TLR4-TIR on the beads, whereas the SARM1-TIR control generated 8-Br-ADPR in the supernatant with higher efficiency.

File: Fig.S9B.zip

Description: 

This archive contains UPLC–HRMS full-scan MS (MS1) data in .mzML format for the samples shown in the corresponding figure panels of the associated research article. ADPR std.mzML contains data for the ADPR reference standard. Myd88.mzML and TIRAP.mzML contain data for the individual MyD88 and TIRAP adaptor protein samples, respectively. TLR1-TLR2.mzML, TLR2-TLR6.mzML, TLR4.mzML, TLR5.mzML, TLR7.mzML, and TLR8.mzML contain data for TIR complexes comprising the indicated TLR or TLR pair together with TIRAP–MyD88 or MyD88 alone, as specified in the corresponding figure panels.

Extracted ion chromatograms (EICs) at m/z 558.0644 were used to assess ADPR bound to the TLR–adaptor TIR complexes, with the ADPR standard serving as the reference. ADPR was also detected in the TLR1–TLR2–TIRAP–MyD88 and TLR2–TLR6–TIRAP–MyD88 TIR complexes.

File: Fig.S10C.zip

Description: 

This archive contains UPLC–HRMS full-scan MS (MS1) data in .mzML format for the ADPR reference standard and the purified proteins and protein complexes shown in Fig.S10B. ADPR std.mzML contains data for the ADPR standard. R808D.mzML, TRAM.mzML, and TRIF.mzML contain data for the individual TLR4(R808D) mutant, TRAM, and TRIF protein samples, respectively. R808D-TIRAP-MYD88.mzML and R808D-TRAM-TRIF.mzML contain data for the TLR4(R808D)–TIRAP–MyD88 and TLR4(R808D)–TRAM–TRIF ternary complexes, respectively.

ADPR associated with the purified proteins or protein complexes was assessed using extracted ion chromatograms (EICs) for the ion at m/z 558.0644 (z = −1), with the ADPR standard as a reference. The purified TLR4(R808D)–TRAM–TRIF TIR complex contained trapped ADPR.

File: Fig.S11C_and_S11D.zip

Description: 

This archive contains UPLC–HRMS full-scan MS (MS1) data in .mzML format for Fig.S11C and S11D. Br-ADPR-standard.mzML contains the data for the 8-Br-ADPR reference standard. All experimental samples were analyzed in the presence of 8-Br-NAD⁺. The buffer control files are Buffer+Br-nad_1.mzML to Buffer+Br-nad_3.mzML. The SARM1, TIRAP, and TLR4 files are Sarm1+Br-nad_1.mzML to Sarm1+Br-nad_3.mzML, TIRAP+Br-nad_1.mzML to TIRAP+Br-nad_3.mzML, and TLR4+Br-nad_1.mzML to TLR4+Br-nad_3.mzML, respectively. The TLR4–TIRAP–MyD88 ternary complex files are TLR4-TIRAP-Myd88+Br-nad_1.mzML to TLR4-TIRAP-Myd88+Br-nad_3.mzML. The suffixes _1, _2, and _3 identify replicate samples within each condition.

Panel C shows extracted ion chromatograms (EICs) for 8-Br-ADPR at m/z 635.9749 in the bead samples collected after the reaction, with the 8-Br-ADPR standard as the reference. Panel D shows the 8-Br-ADPR levels quantified from the EICs in panel C. No 8-Br-ADPR product was detected in the bead sample containing the TLR4–TIRAP–MyD88 TIR complex, suggesting that the complex had no detectable activity toward 8-Br-NAD⁺ under the assay conditions.

File: Fig.S20A.zip

Description: 

This archive contains UPLC–HRMS full-scan MS (MS1) data in .mzML format from the assay of EcRppH activity toward free ADPR. Buffer+ADPR.mzML contains the MS1 data for the buffer control containing ADPR. ECRPPH+ADPR.mzML contains the MS1 data for the reaction in which recombinantly expressed and purified EcRppH (10 µM) was incubated with 1 mM ADPR at 37 °C for 1 h. Targeted extracted ion chromatograms (EICs) for ADPR at m/z 558.0644 are shown for both samples.

File: Fig.S20C.zip

Description: 

This archive contains UPLC–HRMS full-scan MS (MS1) data in .mzML format for the bead-retained TLR4–TIRAP–MyD88 ternary complexes shown in Fig.S20B and the ADPR reference standard. TLR4-TIRAP-Myd88.mzML contains data for the untreated complex, and TLR4-TIRAP-Myd88+Ecrpph.mzML contains data for the EcRppH-treated complex. adpr.mzML contains data for the ADPR reference standard.

ADPR extracted from the bead-retained complexes was monitored using targeted extracted ion chromatograms (EICs) at m/z 558.0644 ([M − H]⁻), with the ADPR standard as the reference. EcRppH treatment did not reduce the complex-associated ADPR signal, and the complex remained assembled. These observations indicate that the buried ADPR was sterically protected from hydrolysis by EcRppH.

File: Fig.S21B-G.zip

Description: 

This archive contains UPLC–HRMS full-scan MS (MS1) data in .mzML format for Fig.S21B−G. Wild-type TLR4-TIR and the E748Q and H706D mutants were incubated with NAD⁺ using the in-bead NADase assay described in Fig. 2B. The files adpr.mzML and 2cadpr.mzML contain data for the ADPR and 2′cADPR reference standards, respectively.

In the sample filenames, -B- identifies the protein-bound bead fraction, and -f- identifies the corresponding protein-free flow-through (supernatant). The suffixes -1 through -4 identify four replicates per condition. The control files are ctrl-B-1.mzML through ctrl-B-4.mzML and ctrl-f-1.mzML through ctrl-f-4.mzML. The wild-type TLR4 files are tlr4-B-1.mzML through tlr4-B-4.mzML and tlr4-f-1.mzML through tlr4-f-4.mzML. The E748Q mutant files are e748q-B-1.mzML through e748q-B-4.mzML and e748q-f-1.mzML through e748q-f-4.mzML. The H706D mutant files are H706D-B-1.mzML through H706D-B-4.mzML and H706D-f-1.mzML through H706D-f-4.mzML.

Panels B and C show targeted extracted ion chromatograms (EICs) for ADPR at m/z 558.0644 (z = −1) in the protein-free supernatant and on the protein-bound beads, respectively, using the ADPR standard as the reference. Panel D quantifies ADPR in both fractions. The H706D mutant did not increase ADPR levels in the supernatant. As with wild-type TLR4-TIR, the ADPR produced by the H706D mutant remained associated with the beads.

Panels E and F show targeted EICs for 2′cADPR at m/z 540.0538 (z = −1) in the protein-free supernatant and on the protein-bound beads, respectively, using the 2′cADPR standard as the reference. Panel G quantifies 2′cADPR in both fractions. For the H706D mutant, 2′cADPR was detected in the supernatant and was undetectable on the beads. The release of 2′cADPR into the supernatant indicates that H706D partially relieves the single-turnover block. These findings support a role for His706 in the TLR4–TIRAP catalytic pocket in favoring linear ADPR production and retaining the product during single-turnover catalysis.

Code/software

Initial mass spectrometry data acquisition and processing were performed using Agilent MassHunter Workstation Software (Qualitative Navigator, version B.08.00). Raw mass spectrometry data are provided in the mzML format here, which can be imported into certain free/open software (such as the "Skyline", https://skyline.ms/project/home/software/Skyline/begin.view), for extracted ion chromatogram (EIC) extraction, peak-area integration, and inspection of MS1 and MS/MS spectra. No custom code, scripts, or bespoke software were developed or used in this study.

Access information

Other publicly accessible locations of the data:

• N/A

Data was derived from the following sources:

• N/A