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M. Dantus and C.L. Kalcic
[51]. Radical-driven sequence ions (a, c, x, and z-ions) were observed for each phosphopeptide, and there was no dominant phosphate loss or phosphate group scrambling. The phosphorylation sites were characterized unambiguously from the fs-LID
spectra [51]. Fs-LID has also been used for the analysis of protonated biomolecules
other than peptides. The method works to dissociate fatty acid chains in lipids [52]
and induce cross-ring cleavages in carbohydrate-based metabolites [53]. The technique was demonstrated to cleave the S–S bond in Arg 8 -vasopressin, eliminating the
need for wet chemistry prior to MS/MS analysis of peptides with strong disulfide
bridges [54].
The most widely adopted ion activation method for MS/MS experiments is collision induced dissociation (CID), where collisions of the precursor ions with a helium bath gas causes fragmentation. As the energy gained through collisions is redistributed throughout the precursor ion, bond cleavage occurs according to bond
dissociation energy. Therefore, the most abundant product ions in the MS/MS spectrum are those formed through the cleavage of the most labile bond or bonds in the
analyte. For peptides, the amino acid composition greatly influences the observed
dissociation pattern by affecting the amenability of the molecule to protonation, the
most likely protonation sites, and proton mobility in the gas phase. These factors can
influence the observed dissociation patterns by enhancing cleavage of specific bonds
[55, 56]. For example, under mobile proton conditions, the backbone heteroatoms
become protonated, making the peptide bonds labile [57]. This makes CID MS/MS
spectra ideal for peptide sequencing when mobile protons are present, as a distribution of peptide bonds between each amino acid along the backbone chain dissociate.
The mass-to-charge ratio of neighboring product ions in the MS/MS spectrum will
differ by the mass of a single amino acid residue, allowing for reconstruction of the
original sequence one amino acid at a time. However, this procedure is interrupted
when unusually labile or non-labile bonds interfere with the standard dissociation
patterns. Under non-mobile proton conditions, the proton or protons are typically
sequestered at basic residues, reducing the observed sequence coverage by CID
[58]. Another obstacle to sequencing are non-labile disulfide bonds between cysteine residues because they give some peptides a cyclic structure. In these cases, a
single peptide bond cleavages fails to fragment the ion, as the two pieces remain
linked at the disulfide bridge and the product is detected at the same m/z value as
the precursor ion. For this reason, peptide samples known to contain S–S bonds are
often chemically reduced prior to MS/MS analysis by CID [59]. While this retains
complete or nearly complete sequence coverage, important structural information
related to sulfur-sulfur connectivity in the native structure is lost. The presence of
a labile chemical modification can interfere with peptide sequencing in a different
manner. For example, during post-translational processing, a protein may become
phosphorylated as part of a cell signaling regulation pathway. Under partially or
non-mobile proton conditions, the covalent bond between the phosphate group and
the amino acid side chain is more labile than the backbone peptide bonds. The Hbonding character of the phosphate group promotes proton transfer from basic side
chains, leading to a charge-directed loss of H 3 PO 4 [60]. This explains why, upon
activation by CID, the phosphate group or groups are cleaved more readily than the
M. Dantus and C.L. Kalcic
[51]. Radical-driven sequence ions (a, c, x, and z-ions) were observed for each phosphopeptide, and there was no dominant phosphate loss or phosphate group scrambling. The phosphorylation sites were characterized unambiguously from the fs-LID
spectra [51]. Fs-LID has also been used for the analysis of protonated biomolecules
other than peptides. The method works to dissociate fatty acid chains in lipids [52]
and induce cross-ring cleavages in carbohydrate-based metabolites [53]. The technique was demonstrated to cleave the S–S bond in Arg 8 -vasopressin, eliminating the
need for wet chemistry prior to MS/MS analysis of peptides with strong disulfide
bridges [54].
The most widely adopted ion activation method for MS/MS experiments is collision induced dissociation (CID), where collisions of the precursor ions with a helium bath gas causes fragmentation. As the energy gained through collisions is redistributed throughout the precursor ion, bond cleavage occurs according to bond
dissociation energy. Therefore, the most abundant product ions in the MS/MS spectrum are those formed through the cleavage of the most labile bond or bonds in the
analyte. For peptides, the amino acid composition greatly influences the observed
dissociation pattern by affecting the amenability of the molecule to protonation, the
most likely protonation sites, and proton mobility in the gas phase. These factors can
influence the observed dissociation patterns by enhancing cleavage of specific bonds
[55, 56]. For example, under mobile proton conditions, the backbone heteroatoms
become protonated, making the peptide bonds labile [57]. This makes CID MS/MS
spectra ideal for peptide sequencing when mobile protons are present, as a distribution of peptide bonds between each amino acid along the backbone chain dissociate.
The mass-to-charge ratio of neighboring product ions in the MS/MS spectrum will
differ by the mass of a single amino acid residue, allowing for reconstruction of the
original sequence one amino acid at a time. However, this procedure is interrupted
when unusually labile or non-labile bonds interfere with the standard dissociation
patterns. Under non-mobile proton conditions, the proton or protons are typically
sequestered at basic residues, reducing the observed sequence coverage by CID
[58]. Another obstacle to sequencing are non-labile disulfide bonds between cysteine residues because they give some peptides a cyclic structure. In these cases, a
single peptide bond cleavages fails to fragment the ion, as the two pieces remain
linked at the disulfide bridge and the product is detected at the same m/z value as
the precursor ion. For this reason, peptide samples known to contain S–S bonds are
often chemically reduced prior to MS/MS analysis by CID [59]. While this retains
complete or nearly complete sequence coverage, important structural information
related to sulfur-sulfur connectivity in the native structure is lost. The presence of
a labile chemical modification can interfere with peptide sequencing in a different
manner. For example, during post-translational processing, a protein may become
phosphorylated as part of a cell signaling regulation pathway. Under partially or
non-mobile proton conditions, the covalent bond between the phosphate group and
the amino acid side chain is more labile than the backbone peptide bonds. The Hbonding character of the phosphate group promotes proton transfer from basic side
chains, leading to a charge-directed loss of H 3 PO 4 [60]. This explains why, upon
activation by CID, the phosphate group or groups are cleaved more readily than the