How to Design 1000-Plex Mass Tags Using the Differential Mass Defect

Multiplexing samples in mass-spectrometry-based proteomics has long been accomplished by isotopologues of small molecules. These chemically identical "mass tags" conjugate to peptides to encode samples with different mass offsets for parallel analysis. The current state of the art for multiplexing with nonisobaric mass tags was recently improved from 3-plex to 9-plex, but what is the largest plex size that can be reasonably achieved with current technology? A full answer to this question requires evaluating current mass spectrometry hardware, facets of which have been well investigated by others. However, it may be underappreciated that multiplexing 1000s of samples with mass tags does not actually require 1000s of isotopes or 1000s of synthesis steps to create. Nonintuitively, high-plex mass tags can require relatively few different isotopes. The focus of this exposition is to characterize the potential of the differential mass defect to create tens to over a thousand isotopologues of small molecules and how careful combinations of these small molecules can combinatorially scale the plex size to minimize the number of synthetic steps. Importantly, we show using formulas from publicly reported compounds that plex sizes in the hundreds, an order of magnitude greater than the state of the art, are achievable using molecules comparable in size to existing commercial tags and that going beyond hundreds may require larger molecules. Additionally, we show how plexing with the differential mass defect allows significantly higher-purity yields during reagent creation for a given plex size. In summary, we investigated and present here how the differential mass defect must be utilized to create high-plex mass tags with limited dopable sites.