Influencia de los empleados.
Etapa 4. Retroalimentación del proceso.
3.3 Conclusiones del tercer capítulo.
The spectrum of the quadrupole isolation of the diglycated [M+2 (C2H2O2)+2 H]2+ (m/z732.37680) species, revealed low abundance of a monoglycated [M+C2H2O2+
2 H]2+ (m/z703.37391) ion. The presence of the monoglycated [M+C2H2O2+2 H]2+ ion is attributed to activation of the diglycated precursor ion [M+2 (C2H2O2)+2 H]2+ during quadrupole isolation, which causes loss of one molecule of C2H2O2. Consequently, the quadrupole isolated precursor ion ([M+2 (C2H2O2)+2 H]2+) was further separated from the monoglycated species in the ICR cell by application of the double resonance (DR) experiment during ECD. This experiment allows ejection of the monoglycated [M+C2H2O2+2 H]2+ species while performing ECD.
The DR-ECD spectrum of the diglycated species showed three types of fragment ions (c/z
·
): unmodified, with single modification (addition of one molecule of C2H2O2, and with double modification (addition of two molecules of C2H2O2. In the present chapter these fragment ions are distinguished using a prescribed nomenclature, as follows. Singly-modified fragment ions are identified by adding a double dagger symbol (‡) to the Roepstorff and Biemann’s nomenclature (c/z·
),[47,48]this single modification was as-sociated with the addition of one molecule of C2H2O2(i.e., a net addition of58.00546Da
to the fragment ion mass). Doubly-modified fragment ions are identified by adding the hash symbol (#) to the normalized (c/z
·
) nomenclature; this modification was linked to the addition of two molecules of C2H2O2(i.e., a net addition of 116.01092Dato the fragment ion mass). Finally, for clarity, the unmodified fragment ions are indicated by using the standardc/z·
nomenclature, with no superscript added.[47,48]The DR(703)-ECD(732) spectrum of the diglycated modified ion [M+2 (C2H2O2) +
2 H]2+, in MeOH/H2O solution is shown in figure 6.2. c#
n modified fragment ions in
position 4 to 10 are observed in the spectrum. The monoglycated modifiedc fragment ion in position 2 shows addition of one molecule of C2H2O2 and is represented as c‡2
in figure6.2. Diglycatedz9#
·
radical fragment ion, unmodifiedc,y, fragment ions, andz
·
radical fragment ions are also present in the spectrum. The modifiedcfragment ion in position 3 were not detected due to the presence of proline.[171,272]Figure 6.2: DR(703)-ECD(732) spectrum of the KM-11 peptide of the precursor ion [M+2 (C2H2O2)+2 H]2+ with ejection of [M+C2H2O2+2 H]2+ in MeOH/H2O so- lution. The symbol ? indicates unassigned peak by the current understanding of the fragmentation mechanism. Highlighted modifiedc#,c‡
2, andz #
9
·
fragment ions in the spectrum and unlabelled peaks are assigned in the detailed peak assignment Table D-5 of the appendixD.The presence of the monoglycated modified c‡2 fragment ion could be attributed to frag- mentation of the monoglycated species, which although ejected by the double resonance experiment, could also be formed during ECD. In order to test the provenance of mono- glycated modifyc‡2, c‡4 toc‡10 fragment ions,z‡9
·
radical fragment ions, along with the diglycatedz9#·
radical fragment ion their relative intensities were normalized (see equa- tion (3.1)) and plotted in figure6.3. Furthermore, ECD experiments were performed without double resonance, and the intensities of thec‡nfragment ions (n= 2,4, . . . ,10), were also normalized and plotted in figure 6.3. Comparison of the normalized relative intensity of the modifiedc‡n fragment ion, z#9·
andz9‡·
radical fragment ions, from the ECD(703) and DR(703)-ECD(732) spectra, revealed that the intensity of the mono- glycatedc‡2, z9‡·
radical fragment ion, and diglycatedz9#·
radical fragment ion did not decrease during the DR-ECD experiment. Normally, in a DR-ECD experiment, the de- crease in the normalized relative intensity of a particular fragment ion indicates the parentage of the ejected ion.[228] Thus, as the intensity of the monoglycatedc‡ion and z9‡
·
radical fragment ion did not decrease, it is though that these species are likely to be derived from the diglycated species ([M+2 (C2H2O2)+2 H]2+), and not from the ejected monoglycated species.Figure 6.3: Comparison of the relative intensity of thec‡nfragment ions andz#9
·
, andz9‡radical fragment ions for the ECD(732) and DR(703)-ECD(732) spectra for the KM-11 peptide.
The CAD spectrum presented in figure 6.4, for the diglycated ion ([M+2 (C2H2O2)+
2 H]2+) in MeOH/H2O, shows modifiedb#
n (n = 3−10) with addition of two molecules of
C2H2O2, andb‡2 fragment ions with the addition of one molecule of C2H2O2. Unmodified
y,b,afragment ions, losses of NH3, loss of the modification, and up to three losses of H2O from the charge reduced-species were also observed in the spectrum. The results showed so far indicate that both the lysine, in position 1, and the arginine, in position 3 are modified by glyoxal in the MeOH/H2O solution, withc‡2 and b‡2 fragment ions likely associated to the diglycated species [M+2 (C2H2O2)+2 H]2+.
The KM-11 peptide was also reacted with glyoxal in PBS and the modified diglycated ion [M+2 (C2H2O2)+2 H]2+species was also present. The DR(703)-ECD(732) spectrum of the [M+2 (C2H2O2)+2 H]2+ species, showed in figure6.5, agrees with the previous results. In summary, addition of two molecules of C2H2O2is observed inc4#toc#10,b#3
Figure 6.4: CAD spectrum of the precursor ion [M+2 (C2H2O2)+2 H]2+for the KM-11 peptide in MeOH/H2O solution. The symbol ? indicates unassigned peaks by the current understanding of the fragmentation mechanism. Highlighted modifiedb# fragment ions in the spectrum and unlabelled peaks are assigned in the detailed peak assignment table D-7 of the appendixD.
c‡2 ,b‡2 , andy9‡fragment ions. It seems clear then that the lysine residue (position 1) is modified by one molecule of C2H2O2and the arginine residue (position 3) is modified either by one or two molecules of C2H2O2.
Figure 6.5: DR(703)-ECD(732) spectrum of the precursor ion [M+2 (C2H2O2)+2 H]2+ with ejection of [M+C2H2O2 +2 H]2+ of the KM-11 peptide in PBS solution. The symbol ? indicates unassigned peaks by the current understanding of the fragmentation mechanism. Highlighted modifiedc#,c‡
2, andx #
9
·
fragment ions and unlabelled peaks are assigned in the detailed peak assignment Table D-8 of the supplementary information.Based on these findings, two possible compounds with the same mass could be formed in the KM-11 peptide in both MeOH/H2O and PBS solution. The first compound that represents diglycation was shown in scheme6.1, which has an elemental composition of C67H103N18O17S and is represented as [M+2 (C2H2O2)+2 H]2+with a theoreticalm/z
of 732.37683. This compound correlates well with the observedc‡2,b‡2 fragment ions, and
z9‡
·
radical fragment ion. These fragments as discussed above, are likely to be derived from the diglycated [M+2 (C2H2O2)+2 H]2+species.A second compound shows the addition of two molecules of C2H2O2 at the arginine residue, as in illustrated in scheme6.2. Interestingly, this compound has similar elemen- tal composition and theoreticalm/z as described above (see scheme6.2) and herein is represented by [M+C4H4O4+2 H]2+. This second compound accounts for the presence ofz9#
·
radical fragment ion,y#9 , andy10# fragment ions. Thus, due to the mixed presence of thec‡2,b‡2, andz9‡·
along with the presence ofz9#·
,y9#, andy10# fragment ions is likely that glyoxal is forming with the KM-11 peptide two isomeric compounds with the same mass, which are detected in the mass spectrometer asm/z 732.37682.Scheme 6.2: Illustration of the addition of two molecules of C2H2O2 at the arginine residue. These diglycation is represented by the formation of [M+C4H4O4)+2 H]2+ species.
The [M+C4H4O4) +2 H]2+ species could have the chemical structures proposed in scheme6.3. The structure proposed in scheme6.3a represents the formation of Nδ-[2-
(dihydroxymethyl)-2H,3aH,4H,6aH-[1,3]dioxolo[5,6-d]imidazolin-5-yl]-L-ornithine, herein referred to as glyoxal dimer at the arginine residue. A second structure could be the formation of Schiff base at the arginine residue, as is illustrated in scheme6.3. It is note- worthy that the proposed formation of the glyoxal dimer (scheme 6.3a) is energetically favored over the Schiff base species (scheme6.3), because of the formation of the stable dioxolane ring.[261]
Scheme 6.3: Representation of the proposed species for the KM-11 peptide reacted with glyoxal in MeOH/H2O and in PBS solutions showing addtion of C4H4O4at the arginine residue: a. proposed glyoxal dimer structure formed at the arginine residue (Nδ-[2-
(dihydroxymethyl)-2H,3aH,4H,6aH-[1,3]dioxolo[5,6-d]imidazolin-5-yl]-L-ornithine);b. proposed Schiff base structure.
In contrast the diglycated [M+2 (C2H2O2)+2 H]2+species (scheme 6.1) could have the lysine residue modified by glyoxal crosslinking with the N-terminus as shown scheme6.4a, but also other chemical structures are possible as proposed in scheme6.4b and scheme6.4c. It seems clear then that acetylation at the N-terminus could block the formation of the possible glyoxal crosslinked species. Moreover, if the species proposed in scheme6.1b and scheme6.4c are formed, acetylation at the N-terminus should not affect the fragmentation pattern and in particularc‡2, andb‡2 fragment ions should be present in the MS/MS experiments. Thus, further experiments were carried out in order to differentiate the proposed intramolecular crosslinking (scheme6.4a) from the Schiff base formation (scheme6.4b) or formation of other species (scheme6.4c) at the lysine
residue, using an acetylated version of the KM-11 peptide (AcKM-11).
Scheme 6.4: Illustration of the possible structures formed at the lysine residue for the KM-11 peptide reacted with glyoxal in MeOH/H2O and in PBS solutions a. Lysine residue crosslinked with glyoxal and the N−terminus ; b. Schiff base formed at the lysine residue;b. heterocyclic amine.