In Q Exactive HF instruments, peptide fragmentation is induce by collision with nitrogen gas, a process referred to as higher energy dissociation (HCD). This type of fragmentation preferentially breaks the peptide at the peptide bond. The intensity is optimized to break each peptide exactly once, resulting in a series of b- and y- ions harboring the n- and c-terminus of the precursor peptide, respectively. From the mass difference between the b- or y-ions the amino acid sequence of the peptide is determined.
Secondary fragmentation can also occur within the amino acid side chains or within molecules attached during posttranslationale modifications (PTMs). This results in diagnostic fragment ions which can be informative for the interpretation of MS2 spectra.