Structural characterization of peptides and peptide fragment ions using high resolution mass spectrometry and ion mobility spectrometry.

Modern mass spectrometers are among the most sensitive and suitable analytical tools for high-throughput protein characterization in proteomics. However, fragment ion rearrangements during gas-phase dissociation processes can limit the success of mass spectrometry (MS) based proteomics approaches. State-of-the-art MS and ion mobility (IM) spectrometry techniques were utilized to further our understanding of gas-phase peptide fragment ion rearrangements. Gas-phase hydrogen/deuterium exchange (HDX) and IM-MS were used to assess the influence of basic histidine residue position and fragment ion size on the observed structural rearrangements. A systematic study of HDX patterns and reaction kinetics for fragments from seven isomeric (Histidine)(Alanine) ₆-NH₂ heptapeptides suggested the presence of at least two ion population types for bn ions (fragment ions formed by peptide amide bond cleavings where charge is kept on the n-terminus side), regardless of the histidine position. Furthermore, IM-MS measurements confirmed the presence of more than one bn isomers for (Histidine)(Alanine)₆-NH₂ fragment ions. Tandem MS and isotope labelings were utilized to investigate possible pathways for generation of structural isomers of bn⁺ fragments. Results from tandem MS of an isotopically labeled (Histidine)(Alanine) ₆-NH₂ peptide revealed the first experimental evidence for generation of sequence-scrambled fragments from y-type ions (where charge remains on the c-terminus side fragment). Thorough analysis of thirty-two additional y-type fragment ions with different charge states (+1 to +3) and sizes (3 to 12 amino acids) confirmed our initial observation of sequence-scrambling from y-type ions (16 out of 32 or ~50 %). Although gas-phase HDX reactions provide valuable structural information, competing reaction channels such as HDX reagent adduct formation can complicate the interpretation of HDX data. We used ab initio calculations combined with HDX, IM-MS, and isotope labeling to identify potential peptide functional groups involved in gas-phase HDX adduct formation. We used benzyloxycarbonyl (Z)-capped dipeptide containing glycine (G) and proline (P) (Z-PG) as a model and studied the influence of protonation and metal ion (Na⁺, K⁺, and Cs⁺) complexation on gas-phase ND₃ adduct formation. Both experimental and theoretical findings indicated that simultaneous availability of carbonyl groups from glycine, proline, and Z were necessary for ND₃ adduct formation with Z-PG.