27-Plex Tandem Mass Tag Mass Spectrometry for Profiling Brain Proteome in Alzheimer’s Disease

Multiplexed isobaric labeling methods, such as tandem mass tags (TMT), remarkably improve the throughput of quantitative mass spectrometry. Here, we present a 27-plex TMT method coupled with two-dimensional liquid chromatography (LC/LC) for extensive peptide fractionation and high-resolution tandem...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2020-05, Vol.92 (10), p.7162-7170
Main Authors: Wang, Zhen, Yu, Kaiwen, Tan, Haiyan, Wu, Zhiping, Cho, Ji-Hoon, Han, Xian, Sun, Huan, Beach, Thomas G, Peng, Junmin
Format: Article
Language:English
Subjects:
Alzheimer Disease - diagnosis
Alzheimer's disease
Amyloidosis
Analytical chemistry
Brain
Chemistry
Chromatography, Liquid
Desalination
Fractionation
Frontal Lobe - chemistry
Humans
Hydrophobicity
Labelling
Liquid chromatography
Mass spectrometry
Mass spectroscopy
Methods
Mitochondria
Multiplexing
Neurodegenerative diseases
Peptides
Peptides - analysis
Proteins
Proteome - analysis
Proteomes
Proteomics
Scientific imaging
Spectroscopy
Tandem Mass Spectrometry
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Summary:Multiplexed isobaric labeling methods, such as tandem mass tags (TMT), remarkably improve the throughput of quantitative mass spectrometry. Here, we present a 27-plex TMT method coupled with two-dimensional liquid chromatography (LC/LC) for extensive peptide fractionation and high-resolution tandem mass spectrometry (MS/MS) for peptide quantification and then apply the method to profile the complex human brain proteome of Alzheimer’s disease (AD). The 27-plex method combines multiplexed capacities of the 11-plex and the 16-plex TMT, as the peptides labeled by the two TMT sets display different mass and hydrophobicity, which can be well separated in LC-MS/MS. We first systematically optimized the protocol for the newly developed 16-plex TMT, including labeling reaction, desalting, and MS conditions, and then directly compared the 11-plex and 16-plex methods by analyzing the same human AD samples. Both methods yielded similar proteome coverage, analyzing >100 000 peptides in >10 000 human proteins. Furthermore, the 11-plex and 16-plex samples were mixed for a 27-plex assay, resulting in more than 8000 protein measurements within the same MS time. The 27-plex results are highly consistent with those of the individual 11-plex and 16-plex TMT analyses. We also used these proteomics data sets to compare the AD brain with the nondementia controls, discovering major AD-related proteins and revealing numerous novel protein alterations enriched in the pathways of amyloidosis, immunity, mitochondrial, and synaptic functions. Overall, our data strongly demonstrate that this new 27-plex strategy is highly feasible for routine large-scale proteomic analysis.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.0c00655