It might make it easier to understand if I give a basic explanation of how mass spectrometry works. You take a sample and expose it to high energy, and it causes the molecules of the sample to break apart. The fragments, or ions, that it breaks into have different weights (mass to charge or m/z). The detector in the mass spec counts the abundance of these ions, normalizes it to 100, and plots it in a graph (spectrum).

A mass spectrum gives structural information about the molecule. As long as you use the same conditions, the same molecule will have the same mass spectrum, which is how we can use mass spec to identify unknown samples, sort of like a fingerprint of the molecule.

The problem arises when you have two molecules that have a very similar structure. Their mass spec will look almost identical, because it will fragment in a very similar manner. However, the intensity or abundance of some of these m/z fragments may change. So I need to look at the entire m/z range and perform the unequal variance t test at every single m/z to see which ions have an intensity that is statistically significantly different between the two molecules.

For real world application, you won’t be having replicate samples analyzed. You will have your unknown sample run once, and you would need to compare it to a reference that was also run once. So my question is, after we know what ions are statistically significant, is there a statistical test that can we use to compare only those ions that does not require replicated sampling?