Peptide sequence analysis plus#
Peptide sequence analysis series#

Peptides containing phosphorylated Ser or Thr residues are examples of the latter case. CAD often fails in this regard when the peptide contains ( i) multiple Arg residues, which inhibit random protonation along the peptide backbone, or ( ii) a PTM that dissociates by a lower energy pathway than that involved in cleavage of the amide linkage. Successful peptide identification by either ECD or CAD can be achieved only when product ions from a complete or nearly complete distribution of amide backbone cleavages are observed in the corresponding tandem MS (MS/MS) spectrum.

Peptide sequence analysis series#

Again, mass differences observed between homologous members of an ion series allow assignment of a particular amino acid to the extra residue in the larger fragment and thus facilitate peptide sequence analysis.įragmentation scheme for production of b- and y-type ions by CAD of a multiply protonated peptide. 2, fragmentation of protonated amide bonds affords a homologous series of complementary product ions of types b and y. Fragment ions are formed when the internal energy of the ion exceeds the activation barrier required for a particular bond cleavage. During each collision, imparted translational energy is converted to vibrational energy that then is rapidly distributed throughout all covalent bonds (picosecond time scale). In this process, peptides that are protonated more or less randomly on backbone amide nitrogen atoms are kinetically excited and undergo collisions with an inert gas such as helium or argon. Ion fragmentation for peptide and protein sequence analysis, with QIT, quadrupole time-of-flight, and QLT instruments, is presently performed with some form of collision-activated dissociation (CAD). As a result, ECD remains a technique exclusively used with FTICR, the most expensive type of MS instrumentation. Despite proposals to circumvent this difficulty ( 29– 31), none have been implemented to date. Thermal electrons introduced into the RF fields of RF 3D quadrupole ion trap (QIT), quadrupole time-of-flight, or RF linear 2D quadrupole ion trap (QLT) instruments maintain their thermal energy only for a fraction of a microsecond and are not trapped. Emulating these conditions in the instruments used most commonly for peptide and protein analyses, those that trap ions with radio frequency (RF) electrostatic fields rather than with static magnetic and electric fields, remains technically challenging. Unfortunately, ECD in its most efficient form requires that the precursor sample ions be immersed in a dense population of near-thermal electrons. The complete amino acid sequence of a peptide is deduced by extending this process to all homologous pairs of fragments within a particular ion series.įragmentation scheme for production of c- and z-type ions after reaction of a low-energy electron with a multiply protonated peptide.īecause ECD occurs along the peptide backbone in a sequence-independent manner, preserves posttranslational modifications (PTMs) ( 7– 14), and can be implemented on a millisecond time scale with precursor-to-product ion conversion efficiencies that approach 30% ( 15– 21), it has become the technique of choice for the analysis of peptide and proteins with FTICR mass spectrometers ( 22– 28). Subtraction of the m/ z values for the fragments within a given ion series that differ by a single amino acid affords the mass and thus the identity of the extra residue in the larger of the two fragments.

Peptide sequence analysis plus#

to an amide nitrogen, a secondary pathway, leads to the formation of carbon monoxide plus a homologous series of complementary fragment ions of types a and y.to the carbonyl groups of the peptide backbone leads to a homologous series of complementary fragment ions of types c and z.One pathway for this process involves generation of an odd-electron hypervalent species ( ) that dissociates to produce RNH 2 and a hydrogen radical ( 6). Capture of a thermal electron by a protonated peptide is exothermic by ≈6 eV (1 eV = 1.602 × 10 -19 J) and causes the peptide backbone to fragment by a nonergodic process, e.g., one that does not involve intramolecular vibrational energy redistribution ( 2– 5). In this method, multiply protonated peptides or proteins are confined in the Penning trap of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer and exposed to electrons with near-thermal energies. Six years ago, McLafferty and coworkers ( 1) introduced a unique method for peptide/protein ion fragmentation: electron capture dissociation (ECD).