Acetylation of lysine 82 initiates TDP-43 nuclear loss of function by disrupting its nuclear import
Methods completeness
Major revisionpanel verdict · 2026-09-01
Methods Completeness & Reagent Traceability Audit
Triggered Categories
The following checklist categories are triggered by content in the manuscript:
- Antibodies/immunodetection (WB, IF, IP, ELISA)
- Cell lines/primary cells
- Human subjects/clinical (postmortem tissue)
- Chemicals/drugs/dosing
- Oligos/plasmids/constructs
- Mass spectrometry (proteomics)
- Microscopy/imaging
- Cross-cutting items (sample size, statistics, software, data availability)
Findings by Category
1. Antibodies/Immunodetection
Trigger: Extensive use of WB, IF, IP, ELISA, and co-IP throughout.
| Antibody/Application | Vendor | Catalog # | Clone | RRID | Dilution | Host/Clonality | Status |
|---|---|---|---|---|---|---|---|
| Primary antibodies (WB, IF, IP) | Supplementary Table S3 referenced | — | — | — | Stated in S3 | — | Missing |
| Anti-6×His-HRP | Proteintech | HRP-66005 | — | — | 1:4000 | — | Present |
| Anti-rabbit-HRP | Proteintech | SA00001-2 | 1:4000 | — | — | — | Present |
| TDP-43 acetylation (K82) antibodies (3 polyclonal) | Sanyou Inc. | — | — | — | Not stated | Rabbit polyclonal | Missing |
| Lamin B1 (loading control) | — | — | — | — | — | — | Unverifiable |
| GAPDH (loading control) | — | — | — | — | — | — | Unverifiable |
| FUS antibody (co-IP) | — | — | — | — | — | — | Unverifiable |
| Importin-α1, importin-α5, importin-β2 (co-IP) | — | — | — | — | — | — | Unverifiable |
Issues:
- HARD missing: Supplementary Table S3 is referenced for antibody details but is not provided in the manuscript text. The three custom polyclonal antibodies against ac-TDP-43(K82) lack vendor catalog numbers, RRIDs, and working dilutions for WB/IF applications.
- HARD missing: Loading control antibodies (Lamin B1, GAPDH) lack vendor, catalog #, and dilution information.
- HARD missing: Antibodies used in co-IP experiments (TDP-43, FUS, importins) lack complete traceability (vendor, catalog #, dilution, host species/clonality).
- SOFT missing: No RRID identifiers provided for any antibody.
2. Cell Lines/Primary Cells
Trigger: iPSC-derived cortical neurons, SH-SY5Y cells, HEK293T cells.
| Cell Line/Source | RRID/CVCL | Authentication (STR) | Mycoplasma Testing | Media/Supplements | Status |
|---|---|---|---|---|---|
| iPSC (WTC11, NGN2-inducible) | — | — | — | Specified (E8, N2, i3Neuron) | Unverifiable |
| SH-SY5Y | ATCC CRL-2266 | — | — | DMEM/F12 + 10% FBS | Partial |
| HEK293T | ATCC CRL-11268 | — | — | DMEM + 10% FBS | Partial |
Issues:
- HARD missing: iPSC source stated as "kind gift of Michael Ward" with no RRID, authentication status, or mycoplasma testing reported.
- HARD missing: No STR authentication or mycoplasma testing reported for any cell line.
- SOFT present: Media and supplements are specified for iPSC differentiation (detailed protocol provided).
- SOFT missing: Passage numbers not stated for any cell line.
3. Human Subjects/Clinical
Trigger: Postmortem motor cortex from sporadic ALS patients and controls.
| Item | Status | Details |
|---|---|---|
| IRB approval | Present | IRB# 10058 (Benaroya) and IRB# 120056 (UCSD) stated |
| Informed consent | Present | "HIPAA-compliant informed consent" stated |
| Participant demographics | Partial | n=6 sALS, n=4 controls; Supplementary Table S2 referenced but not provided in manuscript |
| Inclusion/exclusion criteria | Missing | Not stated |
| Postmortem interval | Present | "Usually under 6 h" stated |
| Sex, age, disease duration | Unverifiable | Supplementary Table S2 referenced; cannot verify from manuscript alone |
Issues:
- HARD missing: Supplementary Table S2 is referenced for participant demographics but not provided in the manuscript text. Sex, age, disease duration, and ALS phenotype are not stated in the main text.
- HARD missing: Inclusion/exclusion criteria for sALS and control groups not specified.
- SOFT missing: No power calculation or sample-size justification for n=6 sALS, n=4 controls.
- SOFT missing: No statement on whether tissue selection was randomized or blinded.
4. Chemicals/Drugs/Dosing
Trigger: Proteasome inhibitors (BTZ, MG132, MRZ), doxycycline, and other reagents.
| Chemical | Vendor | Catalog # | CAS/Identity | Dose/Concentration | Vehicle | Route/Mode | Status |
|---|---|---|---|---|---|---|---|
| Bortezomib (BTZ) | ApexBio | A2614 | — | 2, 20 nM (varies by assay) | — | Added to culture medium | Present |
| MG132 | Selleckchem | S2619 | — | 100 nM | — | Added to culture medium | Present |
| Marizomib (MRZ) | Selleckchem | S7504 | — | 10 nM | — | Added to culture medium | Present |
| Doxycycline | Sigma-Aldrich | D9891 | — | 2 µg/mL | — | Added to culture medium | Present |
| ROCK inhibitor (Y-27632) | Selleckchem | S1049 | — | 10 µM | — | Added to culture medium | Present |
| Protamine sulfate | — | — | — | 10–50 µg/mL | — | Added to viral supernatant | Unverifiable |
Issues:
- SOFT missing: Vehicle/solvent not stated for proteasome inhibitors (assumed DMSO or aqueous, but not specified).
- SOFT missing: Final concentration of protamine sulfate stated as range (10–50 µg/mL) without justification for variation.
- SOFT missing: Duration of drug exposure varies across experiments (12, 24, 48 hr) but is stated per experiment.
5. Oligos/Plasmids/Constructs
Trigger: Lentiviral vectors, siRNA, PCR primers, synthetic peptides.
| Construct/Oligo Type | Sequence | Source/Addgene # | Validation | Status |
|---|---|---|---|---|
| Lentiviral plasmids (pST001 backbone) | — | "Will be deposited to Addgene at publication" | — | Unverifiable |
| TDP-43 variants (WT, K82Q, K82R, 6KR, 14KR, PY-NLS, etc.) | — | Supplementary Table S1 referenced | — | Unverifiable |
| Human TDP-43 siRNA | — | Not specified | — | Missing |
| Packaging plasmids (pMD2.G, psPAX2) | — | Standard 2nd-generation system; no catalog # | — | Unverifiable |
| qRT-PCR primers/probes | — | Supplementary Table S5 referenced | — | Unverifiable |
| TDP-43 peptides (aa77–110, with/without acetylation) | — | "Synthesised by Sanyou Inc." | — | Unverifiable |
Issues:
- HARD missing: Lentiviral plasmids stated to be "deposited to Addgene at publication" but are not yet available; Supplementary Table S1 referenced but not provided in manuscript.
- HARD missing: siRNA target sequence(s) not stated. Only "human TDP-43 siRNAs" mentioned; no sequence, vendor, or catalog # provided.
- HARD missing: qRT-PCR primer and probe sequences not provided in manuscript (Supplementary Table S5 referenced but not included).
- HARD missing: Synthetic peptide sequences and acetylation sites stated generically (e.g., "TDP-43aa77–110 with acetylation at K79, K82, or K84") but exact sequences and synthesis vendor details not fully specified.
- SOFT missing: No off-target assessment for siRNA.
- SOFT missing: Packaging plasmid sources (pMD2.G, psPAX2) not cited; assumed standard but unverified.
6. Mass Spectrometry (Proteomics)
Trigger: TMT quantitative proteomics (nuclear proteome), PTM detection (acetylation, ubiquitination, phosphorylation).
6a. Nuclear Proteome (TMT)
| Parameter | Value | Status |
|---|---|---|
| Instrument | Orbitrap Eclipse | Present |
| Acquisition mode | Data-dependent; MS1 (120k res), MS2 (ion trap CID), MS3 (SPS3, 7.5k res) | Present |
| Sample prep | NE-PER nuclear/cytoplasmic extraction | Present |
| Digestion | Trypsin + Lys-C (1 hr pre-digest, 14 hr main) | Present |
| Labeling | TMT six-plex | Present |
| LC system | nLC 1200, 25 cm × 100 µm BEH C18 (1.7 µm) | Present |
| Gradient | 0–25% B (75 min), 25–40% B (30 min), 40–100% B (10 min), hold 100% B (5 min) | Present |
| Search engine | Rawconverter (MS extraction), DTASelect2 (PSM filtering), Census2 (TMT quantification) | Present |
| Database | UniProt human protein database | Partial |
| FDR threshold | ≤1% at PSM level | Present |
| Static modifications | Carbamidomethylation (Cys), TMT on Lys + N-terminus | Present |
| Precursor mass tolerance | 50 ppm | Present |
| Fragment ion tolerance | 500 ppm (CID), 20 ppm (HECD) | Present |
| Minimum peptide length | 6 amino acids | Present |
| Isobaric purity filter | >0.6 | Present |
| Quantification method | Weighted normalization, one-sample t-test (3 forward + 3 reverse labeling groups) | Present |
| Repository accession | — | Missing |
| Replicates (n) | 3 forward + 3 reverse labeling groups (6 total) | Present |
| Peptide coverage (TDP-43) | 98.3% | Present |
Issues:
- HARD missing: UniProt database version/release date not stated.
- HARD missing: No repository accession (ProteomeXchange/PRIDE) provided for raw MS data or processed results.
- SOFT missing: Monoisotopic precursor selection and dynamic exclusion (60 s) mentioned but not fully parameterized (e.g., intensity threshold for selection).
6b. PTM Detection (Acetylation, Ubiquitination, Phosphorylation)
| Parameter | Value | Status |
|---|---|---|
| Instrument | Not explicitly stated; presumed LC-MS/MS | Unverifiable |
| Sample prep | IP with GFP nanobody magnetic beads | Present |
| Digestion | Trypsin and chymotrypsin independently, combined | Present |
| Enrichment | Titanium dioxide chromatography | Present |
| Acquisition mode | Data-dependent or data-independent (DIA) | Unverifiable |
| Search engine | MaxQuant or Proteome Discoverer | Unverifiable |
| Database | — | Missing |
| FDR | — | Missing |
| Modifications searched | Acetylation (K), ubiquitination (K), phosphorylation (S/T) | Partial |
| Tolerances | — | Missing |
| Repository accession | — | Missing |
Issues:
- HARD missing: PTM MS analysis lacks instrument model, acquisition parameters, search engine version, database, FDR threshold, and mass tolerances.
- HARD missing: No repository accession for PTM MS data.
- HARD unverifiable: "Data analysis conducted with software tools (MaxQuant or Proteome Discoverer)" — both tools mentioned without specifying which was used or versions.
- SOFT missing: No statement on number of replicates for PTM analysis.
7. Microscopy/Imaging
Trigger: Confocal microscopy (IF, live-cell imaging), high-content analysis.
| Parameter | Value | Status |
|---|---|---|
| Instrument (fixed IF) | Yokogawa X1 confocal scanhead on Nikon Ti2 | Present |
| Objective | Plan apo lambda 100× oil (NA 1.45) or 60× oil (NA 1.4) | Present |
| Detector | Spinning disk confocal | Present |
| Live-cell imaging instrument | Yokogawa CQ1 benchtop spinning-disk confocal | Present |
| Live-cell objective | ×40 or ×60 dry | Present |
| Fluorophores/markers | Clover (GFP variant), mRuby, DAPI | Present |
| Imaging settings (temperature, CO₂, humidity) | 37°C, 5% CO₂, humidified | Present |
| Image acquisition software | CQ1 software v.1.05.01.02 (live-cell) | Present |
| Analysis software | — | Missing |
| Gating/segmentation strategy | — | Missing |
| Quantification method | Nuclear/cytoplasmic ratio; nuclear vs. whole-cell fluorescence intensity | Partial |
| Number of cells/fields analyzed | — | Missing |
Issues:
- HARD missing: Analysis software for quantifying nuclear/cytoplasmic localization not specified (e.g., Fiji, CellProfiler, Imaris, custom script).
- HARD missing: Gating or segmentation strategy for defining nuclear vs. cytoplasmic regions not described.
- HARD missing: Number of cells analyzed per condition not stated (e.g., "n=50 cells per condition").
- SOFT missing: Laser wavelengths, detector gain, pinhole size, and pixel dwell time not specified.
- SOFT missing: Thresholds for nuclear/cytoplasmic segmentation not stated.
8. Cross-Cutting Items
8a. Sample Size (n) and Replication
| Experiment | n (biological replicates) | n (technical replicates) | What n represents | Status |
|---|---|---|---|---|
| Proteasome activity assay (Fig. 1A) | — | — | — | Missing |
| TDP-43 fractionation (Fig. 1B) | — | — | — | Missing |
| Immunofluorescence (Fig. 1C–E) | — | — | — | Missing |
| TMT proteomics (Fig. 1E–F) | 6 (3 forward + 3 reverse) | — | Labeling groups | Present |
| RT-PCR stathmin-2 (Fig. 1G) | — | 3 | Technical replicates stated | Partial |
| Co-IP experiments (Fig. 2A–B) | — | — | — | Missing |
| Live-cell imaging (Fig. 2D–I) | — | — | — | Missing |
| Peptide-importin-α1 binding assay (Fig. 3F) | — | — | — | Missing |
| Cell fractionation (Fig. 3C–E) | — | — | — | Missing |
| Lysine-to-arginine mutagenesis (Fig. 4A–H) | — | — | — | Missing |
| Postmortem tissue (Fig. 5B–C) | 6 sALS, 4 controls | — | Individual donors | Present |
| Proteasome activity (mouse/human, Fig. S1A) | 3 mice (per age); 6 sALS, 4 controls | — | Individual animals/donors | Partial |
Issues:
- HARD missing: Biological replicates (n) not stated for most cell-based experiments (proteasome assay, fractionation, IF, co-IP, live-cell imaging, mutagenesis).
- HARD missing: Number of cells/fields analyzed per condition not stated for microscopy experiments.
- SOFT missing: Technical replicates stated only for qRT-PCR (n=3); not stated for other assays.
8b. Statistical Tests and Error Bars
| Figure/Test | Statistical Test | Error Bar Representation | Status |
|---|---|---|---|
| Fig. 1A (proteasome activity) | — | — | Missing |
| Fig. 1D (nucleocytoplasmic ratio) | — | — | Missing |
| Fig. 1E (volcano plot) | One-sample two-sided Student's t-test (unadjusted P) | — | Present |
| Fig. 1F (nuclear protein levels) | — | — | Missing |
| Fig. 1G (RT-PCR) | — | — | Missing |
| Fig. 3F (peptide-importin binding) | — | — | Missing |
| Fig. 4E (nuclear TDP-43 quantification) | — | — | Missing |
| Fig. 5B (ac-TDP-43 levels) | — | — | Missing |
| Methods (general) | "Two-tailed Student's t-tests" for two groups; "one-way ANOVA with Tukey's correction" for ≥3 groups; "Chi-squared tests with Yates' correction" | "Error bars represent SEM unless stated otherwise" | Present |
Issues:
- HARD missing: Specific statistical tests not stated for individual figures (e.g., Fig. 1A, 1D, 1F, 1G, 3F, 4E, 5B).
- SOFT present: General statistical approach and error bar representation stated in Methods, but not consistently applied to all figures.
8c. Software, Tools, and Instrument Versions
| Software/Tool | Version | Status |
|---|---|---|
| Rawconverter | — | Missing |
| DTASelect2 | — | Missing |
| Census2 | — | Missing |
| MaxQuant | — | Unverifiable |
| Proteome Discoverer | — | Unverifiable |
| CQ1 (live-cell imaging) | v.1.05.01.02 | Present |
| Prism (statistics) | 8 | Present |
| R (volcano plot) | — | Missing |
| Fiji/ImageJ (microscopy analysis) | — | Missing |
| Custom analysis scripts | — | Missing |
Issues:
- HARD missing: Versions of MS analysis tools (Rawconverter, DTASelect2, Census2) not stated.
- HARD missing: Versions of MaxQuant or Proteome Discoverer not specified (only "one or the other" mentioned).
- HARD missing: R package version for volcano plot generation not stated.
- HARD missing: Microscopy image analysis software and version not specified.
- SOFT missing: No custom code or scripts provided or deposited.
8d. Data Availability
| Data Type | Repository | Accession | Status |
|---|---|---|---|
| Raw MS data (TMT proteomics) | ProteomeXchange/PRIDE | — | Missing |
| Processed proteomics data | — | — | Missing |
| PTM MS data | ProteomeXchange/PRIDE | — | Missing |
| Microscopy images | — | — | Missing |
| Lentiviral plasmids | Addgene | "To be deposited at publication" | Unverifiable |
| Cell lines (iPSC) | — | — | Missing |
Issues:
- HARD missing: No data-availability statement provided. Manuscript does not specify where raw or processed data will be deposited.
- HARD missing: MS data (both TMT and PTM) not deposited in ProteomeXchange/PRIDE or equivalent.
- HARD missing: Lentiviral plasmids stated as "to be deposited" but not yet available; no interim access mechanism provided.
- SOFT missing: Microscopy image datasets not deposited (e.g., OMERO, Zenodo).
8e. Code Availability
| Code/Analysis | Language | Repository | Status |
|---|---|---|---|
| Custom image analysis | — | — | Missing |
| Statistical analysis scripts | — | — | Missing |
| Data processing pipelines | — | — | Missing |
Issues:
- HARD missing: No code availability statement. Custom analysis (e.g., nuclear/cytoplasmic segmentation, quantification) appears to have been performed but no code or pseudocode provided.
Protocol-Provenance Assessment
| Method | Citation | Resolvability | Status |
|---|---|---|---|
| iPSC differentiation | "as previously described" (ref 37) | Fernandopulle et al. 2018 (PMID resolvable) | Delegated-resolvable |
| Nuclear extraction (NE-PER) | Thermo Scientific product manual | Manufacturer protocol | Delegated-resolvable |
| Nucleus/cytoplasmic fractionation | Abcam Nuclear Extraction Kit | Manufacturer protocol | Delegated-resolvable |
| Lentiviral production | "Detailed guides and protocols posted can be found on the Addgene website" | Addgene protocols (resolvable online) | Delegated-resolvable |
| Proteasome activity assay | Promega Proteasome-Glo reagent | Manufacturer protocol | Delegated-resolvable |
| Cell viability assay | CellTiter-Glo (Promega) | Manufacturer protocol | Delegated-resolvable |
| Confocal microscopy | Standard protocols; instrument manuals | Yokogawa/Nikon manuals | Delegated-resolvable |
| Silver staining | ProteoSilver kit (Sigma) | Manufacturer protocol | Delegated-resolvable |
| ELISA-based binding assays | Custom protocol described in Methods | Full description provided | Self-contained |
| TDP-43 peptide-importin-α1 binding assay | Custom protocol described in Methods | Full description provided | Self-contained |
| Generation of ac-TDP-43(K82) antibodies | "Polyclonal antibodies generated by Sanyou Inc." | Vendor-performed service; no external protocol | Delegated-resolvable |
Issues:
- SOFT: Most methods delegated to manufacturer protocols or cited references, which is acceptable for standard techniques.
- SOFT: Custom assays (peptide-importin binding, ELISA) are described in sufficient detail to be self-contained.
- SOFT: No deviations from cited protocols explicitly stated (e.g., "as described, except..."), though some parameter variations are noted (e.g., proteasome inhibitor doses).
Summary of HARD Missing Items
- Antibodies: Vendor, catalog #, dilution, host species/clonality for primary antibodies (Supplementary Table S3 not provided); ac-TDP-43(K82) antibody dilutions not stated.
- Cell lines: No STR authentication or mycoplasma testing for any line; iPSC source lacks RRID.
- Human subjects: Supplementary Table S2 (participant demographics) not provided; inclusion/exclusion criteria not stated.
- Oligos/plasmids: siRNA target sequence(s) not provided; qRT-PCR primer sequences not provided (Supplementary Table S5 not included); lentiviral plasmids not yet deposited to Addgene.
- Mass spectrometry (PTM): Instrument model, acquisition parameters, search engine version, database, FDR, mass tolerances, and repository accession all missing for PTM analysis.
- Microscopy: Analysis software, segmentation strategy, and number of cells analyzed per condition not specified.
- Sample size: Biological replicates (n) not stated for most cell-based experiments; number of cells/fields analyzed not stated for imaging.
- Statistical tests: Specific tests and error bar representations not stated for individual figures.
- Software versions: Versions missing for Rawconverter, DTASelect2, Census2, MaxQuant/Proteome Discoverer, R, and image analysis software.
- Data availability: No statement provided; MS data not deposited; plasmids not yet available.
Summary of SOFT Missing Items
- Cell lines: Passage numbers not stated.
- Human subjects: No power calculation; no randomization/blinding statement for tissue selection.
- Chemicals: Vehicle/solvent not stated for proteasome inhibitors.
- Microscopy: Laser wavelengths, detector gain, pinhole size, pixel dwell time, and segmentation thresholds not specified.
- Mass spectrometry (TMT): UniProt database version not stated; monoisotopic precursor selection intensity threshold not specified.
- Code availability: No custom code or scripts provided or deposited.
Unverifiable Items (Require Author Clarification)
- Supplementary Tables S1, S2, S3, S5: Referenced but not provided in manuscript; cannot verify contents.
- iPSC source: "Kind gift of Michael Ward" — no RRID or authentication status verifiable.
- Loading control antibodies (Lamin B1, GAPDH): Vendor and catalog # not stated; cannot verify.
- Co-IP antibodies (TDP-43, FUS, importins): Vendor, catalog #, dilution not stated; cannot verify.
- PTM MS analysis: "MaxQuant or Proteome Discoverer" — cannot determine which was used or versions.
- Protamine sulfate: Vendor not stated; concentration range (10–50 µg/mL) not justified.
- Lentiviral plasmids: Stated as "to be deposited at publication" but not currently available; cannot verify sequences or construction details.
Conclusion
This manuscript has substantial gaps in methods completeness and reagent traceability, particularly in:
- Antibody specifications (primary antibodies, custom ac-TDP-43 antibodies)
- Supplementary table provision (S1, S2, S3, S5 referenced but not included)
- Mass spectrometry parameters (PTM analysis lacks critical details)
- Sample size and statistical reporting (n values and specific tests missing for most figures)
- Data and code availability (no repository accessions; plasmids not yet deposited)
- Microscopy quantification (analysis software and segmentation strategy not specified)
An independent group would face significant difficulty reproducing this work without substantial author clarification and provision of missing supplementary materials and data.