期刊
ANALYTICAL CHEMISTRY
卷 92, 期 10, 页码 7162-7170出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.0c00655
关键词
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资金
- National Institutes of Health [R01GM114260, R01AG047928, R01AG053987, RF1AG064909, U54NS110435, U24NS072026, P30AG19610]
- Arizona Department of Health Services [211002]
- Arizona Biomedical Research Commission [4001, 0011, 05-901, 1001]
- ALSAC (American Lebanese Syrian Associated Charities)
- NIH Cancer Center Support Grant [P30CA021765]
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.
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