Assembling the evidence…
Amyotrophic lateral sclerosis (ALS) is a fatal, rapidly progressive neurodegenerative disease of motor neurons for which therapeutics are limited. Improved biomarkers are imperative to improve patient care and therapeutic development. Here, we employed 35-plex isobaric tandem mass tag labeling based on isobutyl-proline reporter group (TMTpro) to perform unbiased proteomic analysis of cerebrospinal fluid (CSF) and plasma from control (n= 28, n= 31) and sporadic ALS (sALS) (n= 39, n= 41), from the Target ALS Global Natural History Study (TALS GNHS). We identified 2,875 proteins in CSF and 1,118 proteins in plasma and identified known and novel differentially expressed proteins (DEPs) between controls and sALS, some of which were orthogonally validated using immunoassay. Comparison of TMTpro-MS and Olink proximity extension assay proteomics revealed common and non-overlapping differentially expressed proteins illustrating strengths unique to each platform. This initial cross-sectional proteomic study of biofluids from the TALS GNHS, with unrestricted availability of study results to the research community, highlights the potential of this resource as a potent platform for ALS biomarker discovery.
We identified 2,875 proteins in CSF and 1,118 proteins in plasma and identified known and novel differentially expressed proteins (DEPs) between controls and sALS, some of which were orthogonally validated using immunoassay.
cross-sectional discovery with modest cohorts and validation limited to three selected proteins
Comparison of TMTpro-MS and Olink proximity extension assay proteomics revealed common and non-overlapping differentially expressed proteins illustrating strengths unique to each platform.
different protein libraries and sample subsets limit inference from differential-expression overlap
A total of 149 proteins were increased and 148 proteins decreased in sALS compared with healthy controls.
directly reported FDR-controlled comparison without covariate adjustment
Derived from the full evaluation — not a separate score.
Strengths
TMTpro-MS and Olink were compared on overlapping CSF and plasma samples, allowing direct description of common and non-overlapping detected proteins and DEPs.
↳ Results, “Comparison of 35plex TMTpro-MS to antibody-based proteomics”
The Methods specify immunodepletion, 35-plex labelling, high-pH fractionation, FAIMS-enabled Orbitrap acquisition, Percolator FDR control, pooled-reference normalisation, and BH correction.
↳ Methods, protein preparation through “Statistical analysis”
ELISA testing in a separate cohort reproduced increased CKM and SERPINA3 while openly reporting that CHAD did not replicate.
↳ Results, “ELISA validation of 35plex TMTpro-MS hits,” Figure 5
Limitations
The primary sALS-versus-control analyses use Welch tests without adjustment for available age, sex, site, or clinical variables, limiting disease-specific interpretation of the DEPs.
↳ Methods, “TALS GNHS Cohort” and “Statistical analysis”
DEP totals from different protein libraries and unequal sample subsets are treated as evidence of superior platform sensitivity without a common reference standard.
↳ Results, cross-platform and tissue-enrichment sections; Conclusions, paragraphs 5–6
Only three proteins underwent independent ELISA testing, one did not replicate, and no site-, demographic-, or disease-stage-stratified analyses are reported.
↳ Methods, “Validation Cohort”; Results, “ELISA validation,” Figure 5
The matched-platform profiling and independent ELISA cohort make this a useful discovery resource, and the detailed analytical workflow supports reproducibility. The main methodological constraint is that disease-group comparisons rely on unadjusted Welch tests despite available demographic and clinical variables. Claims of superior platform sensitivity receive less weight because DEP counts arise from different protein libraries and unequal participant subsets rather than a shared reference standard. The paper is therefore most persuasive as a candidate-generation and data-sharing study, not as evidence of clinically qualified biomarkers or general platform superiority.
Nabu’s assessment, alongside the field’s view.
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Confidence mediumThe study contributes a matched-sample comparison of TMTpro-MS and Olink in CSF and plasma, selected independent-cohort ELISA checks, and a shared proteomic resource. Its advance remains candidate-level because the cohorts are modest and only two of three assayed candidates replicated.
“revealed common and non-overlapping differentially expressed proteins”
The laboratory workflow, pooled reference, peptide-level FDR control, missing-data rule, and BH correction are described in detail. The primary comparisons nevertheless rely on unadjusted Welch tests, and exclusion of group-specific detections plus the smaller Olink subset introduces uncertainty about selection and disease attribution.
“Pairwise comparisons between disease and control groups were conducted using Welch’s t-test”
The manuscript follows a coherent sequence from within-platform findings to cross-platform overlap and ELISA validation. Repeated claims of superior sensitivity are stronger than comparisons of DEP counts across unequal panels and sample subsets support.
“TMTpro-MS showed superior sensitivity over Olink”
The Introduction engages established neurofilament, chitinase, TDP-43, and prior proteomic literature, while the Results disclose CHAD non-replication and plasma ratio compression. The conclusions do not trace the implications of unadjusted cross-sectional comparisons, healthy-only controls, or modest samples into the disease-associated biomarker interpretation.
“Further studies are required to ascertain their role in disease”
Caveats4 of 4 checks
Most sample and DEP arithmetic is coherent, but some protein-count descriptions are not clearly reconciled across analysis stages. The conclusions also interpret unequal DEP counts as platform sensitivity despite differing protein libraries and sample subsets.
Ethics approval, consent, study registration, and a named data portal are declared. The supplied article lacks a conflict-of-interest statement and describes access as unrestricted even though users must apply and receive approval.
Flags: 3 declared / 5 total
45 references in manuscript 45 of 45 checkable references found in an index
No retraction notice found in Retraction Watch.
Sources: Retraction Watch ✓
Where this paper’s evidence sits on the path from initial observation to real-world use.
Independent-cohort ELISA replication of CKM and SERPINA3 provides an early bridge toward accessible assays. No clinical-grade assay, diagnostic accuracy, prognostic performance, or treatment-selection utility is demonstrated.
“CKM was confirmed to be elevated”
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