3, 4 Untargeted quantitative MS methods include DDA/DIA-based label-free quantification 5, 6 and stable isotope labeling, such as SILAC, 7 dimethyl labeling, 8 and isobaric labeling strategies (e.g. 1, 2 Shotgun/bottom-up proteomics, based on the analysis of enzymatic peptides instead of intact proteins, takes advantage of the fact that peptides are much more uniform and compatible with liquid chromatography and MS analysis than proteins. Recent advances in mass spectrometry have allowed for near-global proteomic profiling (more than 10,000 proteins) of a variety of biological samples. Thus, this newly developed protocol is of general application for deep proteomics analysis of biological and clinical samples at sub-microgram levels. Importantly, bioinformatics analysis indicated the microgram-scale protocol had adequate sensitivity and reproducibility to detect differentially expressed proteins in disease-related pathways. 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 9,116 and 10,869 proteins, respectively. 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. Here we present a streamlined protocol to quantify more than 9,000 proteins with 0.5 microgram protein per sample by 16-plex TMT coupled with two-dimensional liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS). 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 micrograms protein in starting materials during a standard analysis.

Res mass tagger