First, useable CID data usually can only be obtained from peptides less than 2-3 kDa, and trypsin generally produces peptides of this size. Using high resolution FTMS instrumentation, partial sequences have been deduced from CID spectra of large peptides or proteins, but in most cases the data one obtains for higher mass molecules is a mess. Doubly-charged precursors also fragment such that most of the peptide bonds break with comparable frequency, such that one is more likely to derive a complete sequence. The second reason for using trypsin proteolysis has to do with the desirability of placing basic residues, notably arginine, at the C-terminus of a peptide. It is a general observation in low energy CID that the presence of arginine in the middle of a peptide will often result in the absence of fragmentations at several contiguous peptide bonds adjacent to the arginine.
Trypsin cleaves on the C-terminal side of arginine and lysine.
By putting the basic residues at the C-terminus, peptides fragment in a more predictable manner throughout the length of the peptide. Low energy CID of peptides results in a limited number of fragment ions . Similar observations can sometimes be made for peptides with an N-terminal glutamic acid or peptides rich in serine or threonine. A similar phenomenon may be observed for peptides containing glycine, where the cleavage on the C-terminal side results in ions of reduced abundance. Often the more abundant internal fragment ions contain proline at the N-terminus of the fragment.