Dataset Information

Optimized sample preparation and data analysis for TMT proteomic analysis of cerebrospinal fluid applied to the identification of Alzheimer's disease biomarkers.

ABSTRACT:

Background

Cerebrospinal fluid (CSF) is an important biofluid for biomarkers of neurodegenerative diseases such as Alzheimer's disease (AD). By employing tandem mass tag (TMT) proteomics, thousands of proteins can be quantified simultaneously in large cohorts, making it a powerful tool for biomarker discovery. However, TMT proteomics in CSF is associated with analytical challenges regarding sample preparation and data processing. In this study we address those challenges ranging from data normalization over sample preparation to sample analysis.

Method

Using liquid chromatography coupled to mass-spectrometry (LC-MS), we analyzed TMT multiplex samples consisting of either identical or individual CSF samples, evaluated quantification accuracy and tested the performance of different data normalization approaches. We examined MS2 and MS3 acquisition strategies regarding accuracy of quantification and performed a comparative evaluation of filter-assisted sample preparation (FASP) and an in-solution protocol. Finally, four normalization approaches (median, quantile, Total Peptide Amount, TAMPOR) were applied to the previously published European Medical Information Framework Alzheimer's Disease Multimodal Biomarker Discovery (EMIF-AD MBD) dataset.

Results

The correlation of measured TMT reporter ratios with spiked-in standard peptide amounts was significantly lower for TMT multiplexes composed of individual CSF samples compared with those composed of aliquots of a single CSF pool, demonstrating that the heterogeneous CSF sample composition influences TMT quantitation. Comparison of TMT reporter normalization methods showed that the correlation could be improved by applying median- and quantile-based normalization. The slope was improved by acquiring data in MS3 mode, albeit at the expense of a 29% decrease in the number of identified proteins. FASP and in-solution sample preparation of CSF samples showed a 73% overlap in identified proteins. Finally, using optimized data normalization, we present a list of 64 biomarker candidates (clinical AD vs. controls, p < 0.01) identified in the EMIF-AD cohort.

Conclusion

We have evaluated several analytical aspects of TMT proteomics in CSF. The results of our study provide practical guidelines to improve the accuracy of quantification and aid in the design of sample preparation and analytical protocol. The AD biomarker list extracted from the EMIF-AD cohort can provide a valuable basis for future biomarker studies and help elucidate pathogenic mechanisms in AD.

SUBMITTER: Weiner S

PROVIDER: S-EPMC9107710 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Publications

Optimized sample preparation and data analysis for TMT proteomic analysis of cerebrospinal fluid applied to the identification of Alzheimer's disease biomarkers.

Weiner Sophia S Sauer Mathias M Visser Pieter Jelle PJ Tijms Betty M BM Vorontsov Egor E Blennow Kaj K Zetterberg Henrik H Gobom Johan J

Clinical proteomics 20220514 1

<h4>Background</h4>Cerebrospinal fluid (CSF) is an important biofluid for biomarkers of neurodegenerative diseases such as Alzheimer's disease (AD). By employing tandem mass tag (TMT) proteomics, thousands of proteins can be quantified simultaneously in large cohorts, making it a powerful tool for biomarker discovery. However, TMT proteomics in CSF is associated with analytical challenges regarding sample preparation and data processing. In this study we address those challenges ranging from dat ...[more]

PMID: 35568819

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Project description:Study designProspective study.ObjectiveTo identify proteins with differential expression in the cerebrospinal fluid (CSF) from 15 clinically normal (control) dogs and 15 dogs with cervical spondylomyelopathy (CSM).Summary of background dataCanine CSM is a spontaneous, chronic, compressive cervical myelopathy similar to human cervical spondylotic myelopathy. There is a limited knowledge of the molecular mechanisms underlying these conditions. Differentially expressed CSF proteins may contribute with novel information about the disease pathogenesis in both dogs and humans.MethodsProtein separation was performed with 2-dimensional electrophoresis. A Student t test was used to detect significant differences between groups (P < 0.05). Three comparisons were made: (1) control versus CSM-affected dogs, (2) control versus non-corticosteroid-treated CSM-affected dogs, and (3) non-corticosteroid-treated CSM-affected versus corticosteroid-treated CSM-affected dogs. Protein spots exhibiting at least a statistically significant 1.25-fold change between groups were selected for subsequent identification with capillary-liquid chromatography tandem mass spectrometry.ResultsA total of 96 spots had a significant average change of at least 1.25-fold in 1 of the 3 comparisons. Compared with the CSF of control dogs, CSM-affected dogs demonstrated increased CSF expression of 8 proteins including vitamin D-binding protein, gelsolin, creatine kinase B-type, angiotensinogen, α-2-HS-glycoprotein, SPARC (secreted protein, acidic, rich in cysteine), calsyntenin-1, and complement C3, and decreased expression of pigment epithelium-derived factor, prostaglandin-H2 D-isomerase, apolipoprotein E, and clusterin. In the CSF of CSM-affected dogs, corticosteroid treatment increased the expression of haptoglobin, transthyretin isoform 2, cystatin C-like, apolipoprotein E, and clusterin, and decreased the expression of angiotensinogen, α-2-HS-glycoprotein, and gelsolin.ConclusionMany of the differentially expressed proteins are associated with damaged neural tissue, bone turnover, and/or compromised blood-spinal cord barrier. The knowledge of the protein changes that occur in CSM and upon corticosteroid treatment of CSM-affected patients will aid in further understanding the pathomechanisms underlying this disease.Level of evidenceN/A.

Project description:The present study used comparative proteomic analysis of cerebrospinal fluid (CSF) in amyotrophic lateral sclerosis (ALS) patients in order to identify proteins that may act as diagnostic biomarkers and indicators of the pathogenesis of ALS. This analysis was performed using isobaric tags for relative and absolute quantitation (iTRAQ) technology, coupled with 2-dimensional liquid chromatography/mass spectrometry. Database for Annotation, Visualization and Integrated Discovery software was utilized for bioinformatic analysis of the data. Following this, western blotting was performed in order to examine the expression of 3 candidate proteins in ALS patients compared with healthy individuals [as a normal control (NC) group] or patients with other neurological disease (OND); these proteins were insulin-like growth factor II (IGF-2), glutamate receptor 4 (GRIA4) and leucine-rich α-2-glycoprotein 1 (LRG1). Clinical data, including gender, age, disease duration and ALS functional rating scale (ALSFRS-R) score, were also collected in the ALS patients. Multiple linear regression analysis was performed between the clinical data and the results of western blot analysis. A total of 248 distinct proteins were identified in the ALS and NC groups, amongst which a significant difference could be identified in 35 proteins; of these, 21 proteins were downregulated and 14 were upregulated. These differentially-expressed proteins were thus revealed to be associated with ALS. The western blot analysis confirmed a proportion of the data attained in the iTRAQ analysis, revealing the differential protein expression of IGF-2 and GRIA4 between the ALS and NC groups. IGF-2 was significantly downregulated in ALS patients (P=0.017) and GRIA4 was significantly upregulated (P=0.016). These results were subsequently validated in the 35-patient ALS and OND groups (P=0.002), but no significant difference was identified in LRG1 expression between these groups. GRIA4 protein expression was higher in male than female patients and was positively correlated with the ALSFRS-R score, meaning that GRIA4 expression was negatively correlated with the severity of ALS, while IGF-2 and LRG1 expression did not correlate with any clinical data. The present study thus demonstrated that GRIA4 expression levels, as a marker of severity, may be used as a reference for the timing of treatment, and that IGF-2 may serve as an effective biomarker of ALS progression.

Dataset Information

Optimized sample preparation and data analysis for TMT proteomic analysis of cerebrospinal fluid applied to the identification of Alzheimer's disease biomarkers.

Background

Method

Results

Conclusion

Publications

Optimized sample preparation and data analysis for TMT proteomic analysis of cerebrospinal fluid applied to the identification of Alzheimer's disease biomarkers.

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