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 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.