Peptides and proteins may contain post-translationally modified phosphorylated amino acid residues, in particular phosphorylated serine (pSer), threonine (pThr) and tyrosine (pTyr). Following earlier work by Lehmann et al., the [M–H]− anions of peptides containing pSer and pThr functionality show loss of the elements of H3PO4. This process, illustrated for Ser (and using a model system), is CH3CONH−C(CH2OPO3H2)CONHCH3 → [CH3CONHC(CH2)CONHCH3 (−OPO3H2)] (a) → [CH3CONHC(CH2)CONHCH3–H]− + H3PO4, a process endothermic by 83 kJ mol−1 at the MP2/6-31++G(d,p)//HF/6-31++G(d,p) level of theory. In addition, intermediate (a) may decompose to yield CH3CONHC(CH2)CONHCH3 + H2PO in a process exothermic by 3 kJ mol−1. The barrier to the transition state for these two processes is 49 kJ mol−1. Characteristic cleavages of pSer and pThr are more energetically favourable than the negative ion backbone cleavages of peptides described previously. In contrast, loss of HPO3 from [M–H]− is characteristic of pTyr. The cleavage [NH2CH(CH2-C6H4-OPO3H−)CO2H] → [NH2C(CH2-C6H4-O−)CO2H (HPO3)] (b) → NH2CH(CH2-C6H4-O−)CO2H + HPO3 is endothermic by 318 kJ mol−1 at the HF/6-31+G(d)//AM1 level of theory. In addition, intermediate (b) also yields NH2CH(CH2-C6H4-OH)CO2H + PO (reaction endothermic by 137 kJ mol−1). The two negative ion cleavages of pTyr have a barrier to the transition state of 198 kJ mol−1 (at the HF/6-31+G(d)//AM1 level of theory) comparable with those already reported for negative ion backbone cleavages. Copyright © 2008 John Wiley & Sons, Ltd.