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 non-isobaric 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 under-appreciated that multiplexing 1000s of samples with mass tags does not actually require 1000s of isotopes, or 1000s of synthesis steps to create. Non-intuitively, 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 synthetic steps. Importantly, we show that plex sizes in the hundreds, an order of magnitude greater than state-of-the-art, are achievable using molecules comparable in size to existing commercial tags, and that going beyond hundreds may require larger molecules. Approaches to achieve high-plex proteomics will almost certainly require using the differential mass defect, so we hope this exposition serves to accelerate progress in reagent development to achieve high plex proteomics.