Sequencing of peptides using MS/MS. Peptide fragmentation by collisionally activated dissociation (CAD) during MS/MS⁴⁸  allows the recognition of complete or partial peptide sequence. Following collisional activation, the peptides undergo fragmentation along the backbone or in their side chains giving rise to product ions that can be attributed to the amino acid sequence. The nomenclature for classifying these ions allows the labeling of all the various fragment ions. The 3 possible cleavage points of the peptide backbone are called a_n,b_n, and c_n when the charge is retained at the N-terminal fragment of the peptide, and x_m,y_m, and z_m when the charge is retained by the C-terminal fragment. The subscripts n and m indicate which amide bond is cleaved counting from the N- and C-terminus, respectively, and thus also the number of amino acid residues contained by the fragment ion. Immonium ions and internal fragments are usually labeled with the letter codes of the amino acids (not indicated in this figure). Product ions derived from fragmentation at the side chain of the peptide (labeled d, v, and w) are not discussed here. Panel A illustrates the ES Q-ToF product ion spectrum of [M+2H]²⁺ at 768 m/z, corresponding to fibrinogen αA from rat hepatocytes, following digestion with trypsin, where [M+2H]²⁺ denotes the doubly charged precursor ion selected for fragmentation. An almost complete series of b and y ions was obtained in this analysis resulting in a facile identification of the peptide sequence. Panel B illustrates the application of MS/MS analysis for the search for sites of phosphorylation. The product ion spectrum, recorded in positive mode, was obtained from the 1-dimensional liquid chromatography-tandem electrospray mass spectrometry analysis (LC-MS/MS) of a phosphorylated peptide. The signals between 1200 and 2000 m/z are magnified by a factor of 2.5. The sites of phosphorylation are searched by scanning for loss of phosphate or for characteristic immonium ions (Figure 7). [M+2H]²⁺ denotes the doubly charged precursor ion selected for fragmentation, and the [M+2H]²⁺-H₃PO₄ denotes the characteristic loss of the phosphate moiety from the precursor ion.