Combined Tissue-Fluid Proteomics to Unravel Phenotypic Variability in Amyotrophic Lateral Sclerosis

Leoni, Emanuela; Bremang, Michael; Mitra, Vikram; Zubiri, Irene; Jung, Stephan; Lu, Ching‐Hua; Adiutori, Rocco; Lombardi, Vittoria; Russell, Claire; Koncarevic, Sasa; Ward, Malcolm; Pike, Ian; Malaspina, Andrea

doi:10.1038/s41598-019-40632-4

Cited by 41 publications

(40 citation statements)

References 38 publications

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“…To circumvent this problem, we have previously used low complexity binders for aggregate separation in biofluid, only to observe that the resulting aggregate fraction had a substantially different composition to the one obtained by ultracentrifugation, the method of choice in the work presented here ( 8 ). Our recent use of two separate proteomic workflows to study the immunological response and the plasma/brain proteome in phenotypic variants of ALS allows further interpretation of our data ( 28 ). In this study, we have used the same brain-enhanced TMT proteomics platform to test neuronal-derived proteins expression in whole plasma, and not in the plasma aggregate-fraction only.…”

Section: Discussionmentioning

confidence: 99%

“…The observed lack of similarity in the composition of brain and plasma aggregates may relate to the limits of proteomic techniques, whereby low abundance proteins, such as the brain-derived ones, may be masked by those with high abundance and detection confounded by the presence of post-translational modifications. To address these shortfalls and gather more information on the potential enrichment of brain-derived proteins in circulating aggregates, we have undertaken further proteomics using a TMTcalibrator™ workflow, where brain lysate was used to enhance detection of proteins in CPA ( [28][29][30] ). 4973 brain-derived proteins were identified, including the three neurofilaments (Nf) protein isoforms (Nf Light (NfL), Nf Medium (NfM) and Nf Heavy (NfH)).…”

Section: Tmtcalibrator™: Brain-derived Proteins In Cpamentioning

confidence: 99%

“…We applied different approaches to assess the relevance of different biological terms, based on their over-representation in the subset of regulated proteins ( 28,32 ). Among the biochemical pathways identified, 69 showed a p-value < 0.05.…”

Section: Functional Analysismentioning

confidence: 99%

“…TMTcalibrator™ workflow was developed by Proteome Sciences plc ( 28,29,32 ) to quantify lowabundance peptides and proteins in matrices with a high degree of biological complexity. In this experiment, ALS brain tissue was used as calibrant, lysate of two ALS brain samples mixed 1:1, loaded at high concentration along with 6 ALS patients and 6 HC individuals which were considered as analytical samples ( Figure S1).…”

Section: Tmtcalibrator™mentioning

confidence: 99%

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Analysis of circulating protein aggregates reveals pathological hallmarks of amyotrophic lateral sclerosis

Adiutori

Puentes

Bremang

et al. 2020

Preprint

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Blood-based biomarkers can be informative of brain disorders where protein aggregation play a major role. The proteome of plasma and circulating protein aggregates (CPA) reflect the inflammatory and metabolic state of the organism and can be predictive of system-level and/or organ-specific pathologies. CPA are enriched with heavy chain neurofilaments (NfH), key axonal constituents involved in brain aggregates formation and biomarkers of the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Here we show that CPA and brain protein aggregates (BPA) from ALS differ in protein composition and appear as a combination of electron-dense large globular and small filamentous formations on transmission electron microscopy. CPA are highly enriched with proteins involved in the proteasome and energy metabolism. Compared to the human proteome, proteins within aggregates show distinct and tissue-dependent chemical features of aggregation propensity. The use of a TMTcalibrator™ proteomics workflow with ALS brain as calibrant reveals 4973 brain-derived low-abundance proteins in CPA, including the products of translation of 24 ALS risk genes. 285 of these (5.7%) are regulated in ALS CPA including FUS (p<0.05). CPA from both ALS and healthy controls affect cell viability when testing endothelial and PC12 neuronal cell lines, while CPA from ALS exert a more toxic effect at lower concentrations. The analysis of resistance to protease enzymes hydrolysis indicates an ALS-specific digestion pattern for NfH using enterokinase. This study reveals how peripheral protein aggregates are significantly enriched with brain proteins which are highly representative of ALS pathology and a potential alternative source of biomarkers and therapeutic targets for this incurable disorder.Significance StatementMolecular mechanism of neurodegeneration like protein aggregation are important brain-specific alterations which need to be addressed therapeutically. Recently described fluid biomarkers of neurodegenerative disorders provide means for stratification and monitoring of disease progression. Here we show that circulating protein aggregates are easily accessible in blood and reproduce important features of brain pathology for an incurable disorder like amyotrophic lateral sclerosis. They represent a source of biomarkers and of novel therapeutics for ALS.

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Section: Discussionmentioning

confidence: 99%

Section: Tmtcalibrator™: Brain-derived Proteins In Cpamentioning

confidence: 99%

Section: Functional Analysismentioning

confidence: 99%

Section: Tmtcalibrator™mentioning

confidence: 99%

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Analysis of circulating protein aggregates reveals pathological hallmarks of amyotrophic lateral sclerosis

Adiutori

Puentes

Bremang

et al. 2020

Preprint

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“…A notable advantage of the TMT approach is that many patient samples can be combined to increase the available peptide mass, allowing identi cation of low abundance species, particularly when coupled with 2-dimensional LC separation. Recently, a number of groups have demonstrated a modi ed TMT labeling scheme that utilizes a "boosting" or "carrier" channel that increases the sensitivity for sample limited samples (18)(19)(20)(21). In these approaches, one or more TMT channels are used to label a larger representative sample that is then mixed with the patient samples for analysis.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Differential Peptide Loading on Tandem Mass Tag-Based Proteomic and Phosphoproteomic Data Quality

Sanford

Wang

Hansen

et al. 2020

Preprint

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Background: Global and phosphoproteome profiling has demonstrated great utility for the analysis of clinical specimens. One major barrier to the broad clinical application of proteomic profiling is the large amount of biological material required, particularly for phosphoproteomics—currently on the order of 25 mg wet tissue weight, depending on tissue type. For hematopoietic cancers such as acute myeloid leukemia (AML), the sample requirement is in excess of 10 million (1E7) peripheral blood mononuclear cells (PBMCs). Throughout the course of a prospective study, this requirement will certainly exceed what is obtainable from many of the individual patients/timepoints. For this reason, we were interested in examining the impact of differential peptide loading across multiplex channels on proteomic data quality. Methods: To achieve this, we tested a range of channel loading amounts (20, 40, 100, 200, and 400 μg of tryptic peptides, or approximately the material obtainable from 5E5, 1E6, 2.5E6, 5E6, and 1E7 AML patient cells) to assess proteome coverage, quantification reproducibility and accuracy in experiments utilizing isobaric tandem mass tag (TMT) labeling. Results: As expected, we found that fewer missing values are observed in TMT channels with higher peptide loading amounts compared to those with lower loading. Moreover, channels with lower loading amounts have greater quantitative variability than channels with higher loading amounts. Statistical analysis of the differences in means among the five loading groups showed that the 20 μg loading group was significantly different from the 400 μg loading group. However, no significant differences were detected among the 40, 100, 200 and 400 μg loading groups. Conclusions: These assessment data demonstrate the practical limits of loading differential quantities of peptides across channels in TMT multiplexes, and provide a basis for designing the optimal clinical proteomics study when specimen quantities are limited.

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Biomarkers in cerebrospinal fluid for amyotrophic lateral sclerosis phenotypes

Zhou

Zeng

Liao

et al. 2023

Ann Clin Transl Neurol

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ObjectiveAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease involving both upper and lower motor neurons. The motor phenotypes of ALS are highly clinically heterogeneous, and the underlying mechanisms are poorly understood.MethodsA comparative proteomic analysis was performed in the cerebrospinal fluid (CSF) of bulbar‐onset (BO) and spinal‐onset (SO) ALS patients and controls (n = 14). Five biomarker candidates were selected from a differentially regulated protein pool, and further validation was performed in a larger independent cohort (n = 92) using enzyme‐linked immunosorbent assay (ELISA).ResultsA total of 1732 CSF proteins were identified, and 78 differentially expressed proteins were found among BO‐ALS patients, SO‐ALS patients, and controls. Five promising biomarker candidates were selected for further validation, and lipopolysaccharide‐binding protein (LBP) and HLA class II histocompatibility antigen, DR alpha chain (HLA‐DRA) were validated. CSF LBP levels were increased in ALS patients compared with controls and higher in BO‐ALS versus SO‐ALS. The increased CSF LBP levels were correlated with the revised ALS Functional Scale (ALSFRS‐R) score. CSF HLA‐DRA levels were specifically elevated in BO‐ALS patients, and there was no significant difference between SO‐ALS patients and controls. Increased HLA‐DRA expression was correlated with decreased survival.InterpretationOur data shows that elevated CSF LBP is a good biomarker for ALS and correlates with clinical severity, and increased HLA‐DRA is a specific biomarker for BO‐ALS and may predict short survival. It also suggests that the microglial pathway and HLA‐II‐related adaptive immunity may be differentially involved in ALS phenotypes and may be new therapeutic targets for ALS.

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Combined Tissue-Fluid Proteomics to Unravel Phenotypic Variability in Amyotrophic Lateral Sclerosis

Cited by 41 publications

References 38 publications

Analysis of circulating protein aggregates reveals pathological hallmarks of amyotrophic lateral sclerosis

Analysis of circulating protein aggregates reveals pathological hallmarks of amyotrophic lateral sclerosis

Evaluation of Differential Peptide Loading on Tandem Mass Tag-Based Proteomic and Phosphoproteomic Data Quality

Biomarkers in cerebrospinal fluid for amyotrophic lateral sclerosis phenotypes

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