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)CONHCH(3) --> [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 + H2PO4 - 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 + PO3 - (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.

Negative ion electrospray mass spectra of the peptides produced by tryptic and chymotrypsin digests of bovine insulin, and from the tryptic digest of lysozyme identify at least 80% of the sequences of these proteins. In particular, negative ion mass spectrometry identifies and positions disulfide moieties, and is the method of choice for identifying this post-translational modification in these two proteins. Intramolecular disulfide functionality is identified by the fragmentation [(M - H)(-)- H(2)S(2)](-) in a digest peptide, and CID of that fragment anion provides amino acid sequencing information. Digest peptides containing an intermolecular disulfide structure undergo facile and diagnostic cleavages. Each cleavage produces a peptide fragment from which CID MS/MS data provide sequencing information.

The characteristic fragmentations of a pTyr group in the negative ion electrospray mass spectrum of the [M-H](-) anion of a peptide or protein involve the formation of PO(3) (-) (m/z 79) and the corresponding [(M-H)(-)-HPO(3)](-) species. In some tetrapeptides where pTyr is the third residue, these characteristic anion fragmentations are accompanied by ions corresponding to H(2)PO(4) (-) and [(M-H)(-)-H(3)PO(4)](-) (these are fragmentations normally indicating the presence of pSer or pThr). These product ions are formed by rearrangement processes which involve initial nucleophilic attack of a C-terminal -CO(2) (-) [or -C(==NH)O(-)] group at the phosphorus of the Tyr side chain [an S(N)2(P) reaction]. The rearrangement reactions have been studied by ab initio calculations at the HF/6-31+G(d)//AM1 level of theory. The study suggests the possibility of two processes following the initial S(N)2(P) reaction. In the rearrangement (involving a C-terminal carboxylate anion) with the lower energy reaction profile, the formation of the H(2)PO(4) (-) and [(M-H)(-)-H(3)PO(4)](-) anions is endothermic by 180 and 318 kJ mol(-1), respectively, with a maximum barrier (to a transition state) of 229 kJ mol(-1). The energy required to form H(2)PO(4) (-) by this rearrangement process is (i) more than that necessary to effect the characteristic formation of PO(3) (-) from pTyr, but (ii) comparable with that required to effect the characteristic alpha, beta and gamma backbone cleavages of peptide negative ions.

The following peptides have been examined in this study: GLDFG(OH), caeridin 1.1 [GLLDGLLGLGGL(NH(2))], 11 Ala citropin 1.1 [GLFDVIKKVAAVIGGL(NH(2))], Crinia angiotensin [APGDRIYVHPF(OH)] and their isoAsp isomers. It is not possible to differentiate between Asp- and isoAsp-containing peptides (used in this study) using negative ion electrospray mass spectrometry. This is because the isoAsp residue cleaves to give the same fragment anions as those formed by delta and gamma backbone cleavage of Asp. The isoAsp fragmentations are as follows: RNHCH(CO(2)H)(-)CHCONHR' --> [RNH(-)(HO(2)CCH=CHCONHR')] --> RNH(-)+HO(2)CCH=CHCONHR' and RNHCH(CO(2)H)(-)CHCONHR' --> [RNH(-)(HO(2)CCH=CHCONHR'] --> (-)O(2)CCH=CHCONHR'+RNH(2). Calculations at the HF/6-31+G(d)//AM1 level of theory indicate that the first of these isoAsp cleavage processes is endothermic (by +115 kJ mol(-1)), while the second is exothermic (-85 kJ mol(-1)). The barrier to the highest transition state is 42 kJ mol(-1). No diagnostic cleavage cations were observed in the electrospray mass spectra of the MH(+) ion of the Asp- and isoAsp-containing peptides (used in this study) to allow differentiation between these two amino acid residues.

[M-H](-) anions from small diphosphopeptides (phosphate groups on Ser, Thr or Tyr) show characteristic peaks corresponding to m/z 177 (H(3)P(2)O(7) (-)), 159 (HP(2)O(6) (-)) and sometimes [(M-H)(-)-H(4)P(2)O(7)](-). M/z 177 and m/z 159 are major peaks in the spectra of small peptides with 1,2, 1,3, 1,4, 1,5 and 1,6 diphosphate substitution, which means that the decomposing [M-H](-) anions must have flexible structures in order for the two phosphate groups to interact with each other. Peptides where the two phosphate groups are more than six amino acid residues apart have not been studied. Theoretical calculations indicate that m/z 177 is formed in a strongly exothermic reaction involving facile nucleophilic interaction between the two phosphate groups: m/z 159 is formed by loss of water from energised m/z 177.