Deep Profiling of Microgram-Scale Proteome by Tandem Mass Tag Mass Spectrometry

Tandem
mass tag (TMT)-based mass spectrometry (MS) enables deep
proteomic profiling of more than 10,000 proteins in complex biological
samples but requires up to 100 μg protein in starting materials
during a standard analysis. Here, we present a streamlined protocol
to quantify more than 9000 proteins with 0.5 μg protein per
sample by 16-plex TMT coupled with two-dimensional liquid chromatography
and tandem mass spectrometry (LC/LC–MS/MS). In this protocol,
we optimized multiple conditions to reduce sample loss, including
processing each sample in a single tube to minimize surface adsorption,
increasing digestion enzymes to shorten proteolysis and function as
carriers, eliminating a desalting step between digestion and TMT labeling,
and developing miniaturized basic pH LC for prefractionation. By profiling
16 identical human brain tissue samples of Alzheimer’s disease
(AD), vascular dementia (VaD), and non-dementia controls, we directly
compared this new microgram-scale protocol to the standard-scale protocol,
quantifying 9116 and 10,869 proteins, respectively. Importantly, bioinformatics
analysis indicated that the microgram-scale protocol had adequate
sensitivity and reproducibility to detect differentially expressed
proteins in disease-related pathways. Thus, this newly developed protocol
is of general application for deep proteomics analysis of biological
and clinical samples at sub-microgram levels.