Supplemental Data
Fragmentation of Singly, Doubly and Triply Charged Hydrogen
Deficient Peptide Radical Cations in Infrared Multiphoton
Dissociation and Electron Induced Dissociation
Anastasia Kalli and Sonja Hess
Proteome Exploration Laboratory
Division of Biology
Beckman Institute
California Institute of Technology
Pasadena, CA 91125, USA
1
Table of Content
Page
MS3 CID of Singly, M+•, Doubly, [M + H]2+•, and Triply, [M + 2H]3+•, Charged Hydrogen Deficient
Radical Cations
4
Figure S1
10
Figure S2
11
Figure S3
12
Figure S4
13
Figure S5
14
References
15
Table S1-S9’: Product ions observed following MS3 CID of:
Table S1. M+• precursor ions of angiotensin I
16
Table S2. [M + H]2+• precursor ions of angiotensin I
17
Table S2’. [M + H]2+• precursor ions of angiotensin I, (linear ion trap only)
17
Table S3. [M + 2H]3+• precursor ions of angiotensin I
18
3+•
Table S3’. [M + 2H] precursor ions of angiotensin I (linear ion trap only)
18
Table S4. M+• precursor ions of [Ile7]-angiotensin III
19
Table S4’. M+• precursor ions of [Ile7]-angiotensin III, (linear ion trap only)
19
Table S5. [M + H]2+• precursor ions of [Ile7]-angiotensin III
20
Table S5’. [M + H]2+• precursor ions of [Ile7]-angiotensin III, (linear ion trap only)
20
Table S6. M+• precursor ions of ACTH 1-10
21
2+•
Table S7. [M + H] precursor ions of ACTH 1-10
22
Table S7’. [M + H]2+• precursor ions of ACTH 1-10, (linear ion trap only)
22
Table S8. [M + H]2+• precursor ions of neurotensin
23
Table S8’. [M + H]2+• precursor ions of neurotensin, (linear ion trap only)
23
Table S9. [M + 2H]3+• precursor ions of neurotensin
24
Table S9’. [M + 2H]3+• precursor ions of neurotensin, (linear ion trap only)
25
3
Table S10-S18: Product ions observed following MS IRMPD of:
Table S10. M+• precursor ions of angiotensin I
25
Table S11. [M + H]2+• precursor ions of angiotensin I
26
Table S12. [M + 2H]3+• precursor ions of angiotensin I
27
Table S13. M+• precursor ions of [Ile7]-angiotensin III
28
Table S14. [M + H]2+• precursor ions of [Ile7]-angiotensin III
28
Table S15. M+• precursor ions of ACTH 1-10
29
2+•
Table S16. [M + H] precursor ions of ACTH 1-10
30
Table S17. [M + H]2+• precursor ions of neurotensin
31-32
Table S18. [M + 2H]3+• precursor ions of neurotensin
33-34
Table S19-S27: Product ions observed following MS3 EID of:
Table S19. M+• precursor ions of angiotensin I
35
Table S20. [M + H]2+• precursor ions of angiotensin I
35
3+•
Table S21. [M + 2H] precursor ions of angiotensin I
36
Table S22. M+• precursor ions of [Ile7]-angiotensin III
37
Table S23. [M + H]2+• precursor ions of [Ile7]-angiotensin III
38
Table S24. M+• precursor ions of ACTH 1-10
39
Table S25. [M + H]2+• precursor ions of ACTH 1-10
40
Table S26. [M + H]2+• precursor ions of neurotensin
41-42
3+•
Table S27. [M + 2H] precursor ions of neurotensin
43-44
Table S28-S31: Observed amino acid side chain losses in MS3 CID:
Table S28. From the radical precursor ions of angiotensin I
45
2
Table S29. From the radical precursor ions of [Ile7]-angiotensin III
Table S30. From the radical precursor ions of ACTH
Table S31. From the radical precursor ions of neurotensin
Table S32-S35: Observed amino acid side chain losses in MS3 IRMPD:
Table S32. From the radical precursor ions of angiotensin I
Table S33. From the radical precursor ions of [Ile7]-angiotensin III
Table S34. From the radical precursor ions of ACTH
Table S35. From the radical precursor ions of neurotensin
Table S36-S39: Observed amino acid side chain losses in MS3 EID:
Table S36. From the radical precursor ions of angiotensin I
Table S37. From the radical precursor ions of [Ile7]-angiotensin III
Table S38. From the radical precursor ions of ACTH
Table S39. From the radical precursor ions of neurotensin
45
46
46
47
47
48
48
49
49
50
50
3
MS3 CID of Singly, M+●, Doubly, [M + H]2+●, and Triply, [M + 2H]3+●, Charged Hydrogen Deficient
Radical Cations
All assigned product ions are summarized in Tables S1-S9’ and observed amino acid side chain losses in
Tables S28-S31.
MS3 CID of Angiotensin I
Singly Charged Radical Cations, M+●
For angiotensin I, mainly a-type product ions, CO2 loss and side chain losses were observed (Figure S1).
Formation of a-type product ions has been suggested to be favorable when the charge is located at the Nterminus [1] and it has been observed in a number of CID spectra of peptide radical cations containing an
arginine at or close to the N-terminus [1-5]. For angiotensin I, the arginine is located next to the Nterminus and therefore formation of a-type product ions is favorable. Some z-type product ions were also
observed, but they were detected with lower yields.
Furthermore, enhanced cleavage at aromatic residues was apparent from the formation of abundant a8, a9
and a6 product ions. Formation of a8 and a9 product ions corresponds to cleavage next to histidine at
position 9, and formation of the a6 product ion corresponds to cleavage at the C-terminal side of histidine
at position 6. Formation of the a8 product ion also corresponds to cleavage at the C-terminal side of
phenylalanine. This observation, preferential cleavage at aromatic residues, is in accordance with
previous reports [3-5].
Doubly Charged Radical Cations, [M + H]2+●
Similar to the results obtained for the singly charged species, MS3 CID of the doubly charged radical
precursor ions of angiotensin I, [M + H]2+●, resulted in abundant CO2 loss and formation of a-type
product ions (Figure S1). Side chain loss from tyrosine (106.042 Da) was more prevalent for the doubly
charged radical precursor ions. Side chain losses from arginine (C4H8N3● 86.0718 Da), aspartic acid
(COOH● 44.9976 Da) and leucine (C3H7● 43.0548 Da), which were not observed for the singly charged
species, were present in the spectrum of the doubly charged counterpart. The side chain loss from aspartic
acid was only observed from the product ions. Furthermore, additional backbone bond product ions, not
detected in the spectrum of the singly charged species, were observed for this charge state. These include
b-, y- and several a- and z-type product ions, with the most abundant backbone product ions being a9, a6,
a3, a4 and y4. The y4 product ions was also detected in the CID spectrum of the even electron species (data
4
not shown) indicating that the formation of this product ion is charge driven. For the singly charged
radical cations no b- or y-type product ions were detected.
Triply Charged Radical Cations, [M + 2H]3+●
MS3 CID of triply charged radical cations of angiotensin resulted mainly in CO2 loss, tyrosine side chain
loss (106.0418 Da) and formation of the y4 product ion (Figure S1). The y4 product ion was also detected
as an abundant product ion in the CID spectrum of the triply charged even electron precursor ions (data
not shown), indicating that the formation of this ion is not initiated by a radical driven dissociation. As
expected, the product ions corresponding to side chain losses were absent in the spectrum of the even
electron precursors ions (data not shown). Backbone product ions exhibited extensive COOH● (44.9977
Da) loss from aspartic acid. In addition, several a-, b- and y-type product ions were also present.
When comparing the results obtained for the triply charged radical cations with those obtained for the
singly and doubly charged counterpart, loss of CO2 and tyrosine side chain loss were dominant for all
charge states. For the triply and doubly charged radical cations b- and y- product ions and also COOH•
loss from aspartic acid were detected while these fragmentation pathways were absent for the singly
charged radical cations. Triply charged radical cations exhibited less extensive fragmentation when
compared to their doubly charged counterpart (Scheme 2). For instance, several backbone product ions
such as a5, a6, y2, y8, z3●, z6●, z7● were only detected for the doubly charged species. Furthermore, for the
doubly and triply charged species more backbone product ions were detected, (Scheme 2), when
compared to the number of product ions obtained for the singly charged counterpart.
MS3 CID of [Ile7]-angiotensin III
Singly Charged Radical Cations, M+●
Similar to the results obtained for angiotensin I, MS3 CID of [Ile7]-angiotensin III yielded mainly atype product ions and side chain losses (Figure S2). For [Ile7]-angiotensin III, the arginine is located at the
N-terminus, therefore, formation of a-type product ions is preferable over other dissociation pathways, as
discussed above. Formation of some z- and y-type product ions was also observed, but these product ions
were detected with lower abundances. Dominant cleavage at the C-terminal side of histidine, to produce
the a5 product ion, and at the C-terminal side of tyrosine, to produce the a3 product ion, was also
observed.
5
Doubly Charged Radical Cations, [M + H]2+●
The MS3 CID spectrum of the doubly charged radical cations of [Ile7]-angiotensin III is dominated by
side chain loss from tyrosine (106.042 Da) and arginine (86. 0718 Da) and formation of a2, a3 and [b5 H]+● product ions (Figure S2). The a2 and a3 product ions correspond to cleavage next to tyrosine and the
[b5 - H]+● corresponds to cleavage between histidine and proline. The former two were absent following
CID of the even electron precursor ions and the latter was observed as an even electron species (data not
shown). We can, therefore, conclude that formation of a-type product ions was radical driven, whereas
formation of the [b5 - H]+● product ion was charge driven. In addition to these backbone product ions,
several y- and z-type product ions were also produced. In fact, the z-type product ions were observed with
relatively high yields. Formation of odd electron z-type product ions has been previously observed in MS3
CID of hydrogen deficient radical cations and it was suggested that odd electron z-type product ions can
be formed from unstable [xn + H]+● product ions by loss of isocyanic acid from the latter through a radical
driven process [3]. For the doubly protonated species, CO2 loss from the radical precursor ions was
absent, whereas this loss corresponded to the most abundant product ion in the MS 3 CID spectrum of the
singly charged counterpart. Overall, less side chain losses and more backbone product ions were detected
for the doubly charged radical precursor ions (Figure S2 and Scheme 3). These backbone product ions
originated from both radical and charge driven processes.
MS3 CID of ACTH 1-10
Singly Charged Radical Cations, M+●
For ACTH 1-10 predominant cleavages next to tryptophan were apparent from the formation of abundant
a9, a8 and c8 product ions and abundant side chain loss from tryptophan (Figure S3). Cleavages at the Cterminal side of tyrosine to produce z8● and x8 - H2O product ions and side chain loss from tyrosine were
also detected with high yields. Furthermore, a wide range of product ions including a-, b-, c-, y-, x- and ztype, were detected. This non-specific fragmentation has been previously observed for singly charged
radical cations featuring a basic amino acid residue, Arg or His, at or close to the C-terminus [1-3].
Doubly Charged Radical Cations, [M + H]2+●
We then examined the MS3 CID spectrum of the doubly charged radical cations of ACTH 1-10. The
major fragmentation pathways include cleavage of the tryptophan side chain (C9H7N 129.058 Da), CO2
loss and formation of the c8 product ion, resulting from cleavage at the N-terminal side of tryptophan
(Figure S3). Enhanced cleavage at the C-terminal side of tryptophan was also apparent from the detection
of abundant a9- and [b9- H]● product ions. Two additional product ions, a7 and [z2● - H], corresponding to
cleavage at the C-terminal side of phenylalanine and N-terminal side of tryptophan, respectively, were
6
also detected. Furthermore, secondary product ions resulting from amino acid side chain losses from both
the precursor and product ions were detected with lower yields.
The MS3 CID spectra of the singly and doubly protonated radical precursor ions displayed significant
differences in the relative abundances of the observed product ions (Figure S3). For instance, for the
doubly protonated radical species, the most abundant product ions were those from tryptophan side chain
loss followed by formation of the c8 product ion, which was detected in both singly and doubly protonated
form. For the singly charged counterpart the most abundant product ions were the a 9 and H2O loss from
the radical precursor ions. Methionine side chain losses (C3H6S 74.019 Da, C2H5S● 61.0112 Da) were
significantly more dominant in the CID spectrum of the singly protonated radical cations. In addition,
some product ions were only detected either from the singly or from the doubly charged radical cations.
For example, the z8● and x8 - H2O were detected with high yield for the singly protonated radical cations
but were absent in the spectrum of the doubly protonated radical counterpart. The b9 product ion was
detected as an odd electron species for the doubly charged radical cations and as an even electron species
for the singly charged radical precursor ions. Despite these differences, cleavages associated with the
presence of a tryptophan represented the major fragmentation pathways for both charge states. This
behavior, abundant tryptophan side chain loss and/or favored cleavages next to tryptophan, has been
previously reported in CID of hydrogen deficient species [3, 6, 7] and also in electron-based reactions of
protonated and deprotonated amino acids and peptides [8-11].
MS3 CID of Neurotensin
Doubly Charged Radical Cations, [M + H]2+●
For this peptide cleavage of leucine side chain (C3H7• 43.0548 Da) dominated the spectrum (Figures S4
and S5A). This product ion was detected with three times higher intensity when compared to the other
product ions in the spectrum. Side chain losses of glutamic acid (C2H3O2● 59.0133 Da), lysine (C4H9N
71.0735 Da) and leucine or isoleucine (C4H8 56.0626 Da) were also present. Similar to the other peptides
examined cleavage at the tyrosine residue to produce the a112+ product ion and tyrosine side chain loss
(C7H6O 106.042 Da) from the precursor ions were also abundant. The product ion detected at m/z
757.417 can be assigned as an a12 product ion or as [M● - 59(E) - 99(R)], or [M● - 72(E) - 86(R)]. None of
these assignments can be eliminated because, formation of a-type product ion is a common fragmentation
pathway in CID of hydrogen deficient radical cations, and the specific side chain losses from glutamic
acid, corresponding to 59 Da and 72 Da, and from arginine, corresponding to 99 Da and 86 Da, have also
been observed and reported for peptide radical cations [1, 3]. In fact, it is possible that the observed ion
represents a mixture of these fragmentation pathways. Several other backbone product ions, including a-,
7
y- and z-type, were present but were detected with lower intensities. The y7 product ion corresponding to
cleavage at the N-terminal side of proline was detected as y7 - 2H. These hydrogen deficient y-type
product ions occurring N-terminal to proline have been previously observed in high energy CID [12], in
femtosecond laser-induced ionization/dissociation [13] and electron induced dissociation [14].
Triply Charged Radical Cations, [M + 2H]3+●
Following MS3 CID of the triply charged radical cations of neurotensin abundant product ions
corresponding to tyrosine (C7H6O● 106.0418 Da), glutamic acid (C2H3O2● 59.0133 Da), leucine (C3H7●
43.0548 Da, C4H8 56.0626 Da), arginine (C4H9N3 99.0796 Da, C3H8N3● 86.0718 Da) and lysine (C3H8N●
58.0657 Da, C4H9N 71.0735 Da) side chain losses from the precursor ions were present (Figures S4 and
S5B). A wide range of product ions including a-, b-, y-, c-, x- and z- product ions was detected. From
these, the a11, y7, y10, y11 and z10● were the most abundant. The z10● - 59(E) product ion, detected in both
doubly and triply protonated form, and the a12 - 106(Y) were also detected with high yield. Formation of
the a11 and z10● product ions corresponds to cleavage at the C-terminal side of tyrosine at position 11 and
3, respectively. The y7, y10, and y11 were also present in the spectrum of the triply charged even electron
precursor ions, whereas the a11 and z10● were absent (data not shown). The majority of backbone product
ions exhibited extensive secondary fragmentation by loss of several side chains from their constituent
amino acid residues. This extensive secondary fragmentation was also observed from the precursor ions.
The MS3 CID of the triply charged radical cations of neurotensin displayed several differences when
compared to MS3 CID of the doubly charged species. For the triply charged radical precursor ions, more
extensive fragmentation was observed and a large number of backbone product ions was detected with
high relative intensities. For the doubly charged counterpart the majority of backbone product ions were
detected with very low intensities. For example the z10● product ion was detected with a relative intensity
of 3% and 15% for the doubly and triply charged radical precursor ions, respectively. For the doubly
charged species, the most abundant product ions, with the exception of a11, corresponded to amino side
chain losses. Furthermore, more extensive secondary fragmentation was observed for the triply charged
radical precursor ions. Some backbone product ions were unique for each charge state. For instance, the
a9 and y12 were only detected for the doubly charged species. The y10 and y11, detected with 18% and 29%
relative intensity, respectively, were only present in the spectrum of the triply charged radical precursor
ions. A common fragmentation channel, which was enhanced for both charge states, was cleavage
associated with the presence of tyrosine. Another commonality was the formation of the y7 - 2H product
ion.
8
Conclusions
Based on the fragmentation pathways observed in the MS3 CID spectra of the peptides examined here we
can conclude that for singly charged species radical driven dissociation dominated. MS 3 CID of the [M +
H]2+● precursor ions yielded product ions resulting from both radical and proton driven dissociation,
although product ions corresponding to radical driven processes were detected with higher relative
intensities. For the triply charged radical cations, [M + 2H]3+●, charge driven fragmentation becomes also
favorable. As a result, product ions initiated by radical and charge driven dissociation were detected with
comparable yields. Numerous amino acid side chain losses and small neutral losses, such as CO2 and H2O
losses, were observed for all three singly charged radical cations examined. It should be noted that the
occurrence and the relative abundances of each amino acid side chain loss differ are not identical for each
peptide and each charge state. In general, these side chain losses are in agreement with previous
investigations [1, 3]. The side chain loss from tryptophan corresponding to C8H6N● (116.05 Da) was
observed in our experiments but it has not been previously reported in CID of hydrogen deficient species.
This loss was also observed in ECD [15] and in CID of odd electron z●-type product ions formed in ETD
[16]. Regardless of the precursor ions charge state, a common fragmentation channel observed for all
peptides examined was the enhanced fragmentation at aromatic amino acid residues, i.e. histidine,
tyrosine, tryptophan and phenylalanine, in agreement with previous reports [3-5].
9
Figure S1. Product ion abundance distribution obtained by MS3 CID of angiotensin I. Product ion
abundances were obtained from the ion trap data to account for the time of flight effect. Only the most
abundant product ions are displayed in the Figure. A complete list of all observed product ions is given in
Tables S1-S3’ and all backbone product ions are summarized in Scheme 2.
.
10
Figure S2. Product ion abundance distribution obtained by MS3 CID of [Ile7]-angiotensin III. Product
ions abundances were obtained from the ion trap data to account for the time of flight effect. Only the
major peaks are displayed in the Figure. A complete list of all observed product ions can be found in
Tables S4-S5’ and all backbone product ions are summarized in Scheme 3.
11
Figure S3. Product ion abundance distribution obtained by MS3 CID of ACTH 1-10. Product ions
abundances were obtained from the ion trap data to account for the time of flight effect. Only the most
abundant product ions are shown in the Figure. A complete list of all observed product ions is given in
Tables S6-S7’. All detected backbone product ions are displayed in Scheme 4.
# = more than one assignment possible based on m/z ratio
12
Figure S4. Product ion abundance distribution obtained by MS3 CID of neurotensin. Product ions
abundances were obtained from the ion trap data to account for the time of flight effect. Only the major
peaks are displayed in the Figure. A complete list of all observed product ions can be found in Tables S8S9’and all detected backbone product ions are summarized in Scheme 5.
# = more than one assignment possible based on m/z ratio
13
Figure S5. MS3 CID of (A) doubly and (B) triply charged radical cations of neurotensin, pELYENKPRRPYIL-OH.
The inset B’ (m/z= 400-520) shows the product ions detected only in the linear ion trap. The MS3 CID spectrum of
the doubly charged precursor ions (A) was dominated by leucine side chain loss, whereas for the triply charged
radical precursor ions (B) additional side chain losses and backbone product ions were prevalent. Only the major
peaks are labeled in the Figure. A complete list of all observed product ions is given in Tables S8, S8’, S9 and S9’.
* = noise peak, # = more than one assignment possible based on m/z ratio, ¥ = product ions corresponding to
combined side chain losses.
14
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Laskin, J., Yang, Z. B., Ng, C. M. D., Chu, I. K.: Fragmentation of alpha-Radical Cations of ArginineContaining Peptides. J. Am. Soc. Mass Spectrom. 21, 511-521 (2010)
Laskin, J., Yang, Z. B., Lam, C., Chu, I. K.: Charge-Remote Fragmentation of Odd-Electron Peptide
Ions. Anal. Chem. 79, 6607-6614 (2007)
Sun, Q. Y., Nelson, H., Ly, T., Stoltz, B. M., Julian, R. R.: Side Chain Chemistry Mediates Backbone
Fragmentation in Hydrogen Deficient Peptide Radicals. J. Proteome Res. 8, 958-966 (2009)
Zhang, L. Y., Reilly, J. P.: Radical-Driven Dissociation of Odd-Electron Peptide Radical Ions
Produced in 157 nm Photodissociation. J. Am. Soc. Mass Spectrom. 20, 1378-1390 (2009)
Song, T., Xu, M., Quan, Q., Siu, C., Fang, D., Chu, I. K.: Effect of Basic Residues on Selective C
alpha-C Bond Cleavages of Peptide Radical Cations. Proceedings of the 58th ASMS Conference on
Mass Spectrometry and Allied Topics, Salt Lake City, UT, 2010.
Bagheri-Majdi, E., Ke, Y. Y., Orlova, G., Chu, I. K., Hopkinson, A. C., Siu, K. W. M.: CopperMediated Peptide Radical ions in the Gas Phase. J. Phys. Chem. B 108, 11170-11181 (2004)
Siu, C. K., Ke, Y. Y., Orlova, G., Hopkinson, A. C., Siu, K. W. M.: Dissociation of the N-C-alpha Bond
and Competitive Formation of the [z(n) - H](center dot+) and [c(n)+2H](+) Product Ions in Radical
Peptide Ions Containing Tyrosine and Tryptophan: The Influence of Proton Affinities on Product
Formation. J. Am. Soc. Mass Spectrom. 19, 1799-1807 (2008)
Haselmann, K. F., Budnik, B. A., Kjeldsen, F., Polfer, N. C., Zubarev, R. A.: Can the (M center dotX)Region in Electron Capture Dissociation Provide Reliable Information on Amino Acid
Composition of Polypeptides? Eur. J. Mass Spectrom. 8, 461-469 (2002)
Lioe, H., O'Hair, R. A. J.: Comparison of Collision-Induced Dissociation and Electron-Induced
Dissociation of Singly Protonated Aromatic Amino Acids, Cystine and Related Simple Peptides
Using a Hybrid Linear Ion Trap-FT-ICR Mass Spectrometer. Anal. Bioanal. Chem. 389, 1429-1437
(2007)
Kruger, N. A., Zubarev, R. A., Carpenter, B. K., Kelleher, N. L., Horn, D. M., McLafferty, F. W.:
Electron Capture versus Energetic Dissociation of Protein Ions. Int. J. Mass Spectrom. 182, 1-5
(1999)
Kalli, A., Hakansson, K.: Preferential Cleavage of S-S and C-S Bonds in Electron Detachment
Dissociation and Infrared Multiphoton Dissociation of Disulfide-Linked Peptide Anions. Int. J.
Mass Spectrom. 263, 71-81 (2007)
Medzihradszky, K. F., Burlingame, A. L.: The Advantages and Versatility of a High-Enegy Collison
Induced Dissociation-Based Strategy for the Sequence and Structural Determination of Proteins.
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Smith, S. A., Kalcic, C. L., Safran, K. A., Stemmer, P. M., Dantus, M., Reid, G. E.: Enhanced
Characterization of Singly Protonated Phosphopeptide Ions by Femtosecond Laser-induced
Ionization/Dissociation Tandem Mass Spectrometry (fs-LID-MS/MS). J. Am.Soc. Mass Spectrom.
21, 2031-2040 (2010)
Kalli, A., Grigorean, G., Hakansson, K.: Electron Induced Dissociation of Singly Charged Peptide
Anions. J. Am. Soc. Mass Spectrom. Accepted. (2011)
Savitski, M. M., Nielsen, M. L., Zubarev, R. A.: Side-Chain Losses in Electron Capture Dissociation
to Improve Peptide Identification. Anal.Chem. 79, 2296-2302 (2007)
Han, H., Xia, Y., McLuckey, S. A.: Ion Trap Collisional Activation of c and z(center dot) Ions
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15
Table S1. Product ions observed following MS3 CID of the singly protonated, M+●, species of angiotensin
I, DRVYIHPFHL. The numbers in parenthesis indicate the product ions abundances in the linear ion trap.
All other measurements were made in the FT-ICR cell.
16
Table S2. Product ions observed following MS3 CID of the doubly protonated, [M + H]2+●, species of
angiotensin I, DRVYIHPFHL. The numbers in parenthesis indicate the product ions abundances in the
linear ion trap. All other measurements were made in the FT-ICR cell.
Table S2’. Product ions detected only in the linear ion trap following MS3 CID of the doubly protonated,
[M + H]2+●, species of angiotensin I, DRVYIHPFHL.
17
Table S3. Product ions observed following MS3 CID of the triply protonated, [M + 2H]3+●, species of
angiotensin I, DRVYIHPFHL. The numbers in parenthesis indicate the product ions abundances in the
linear ion trap. All other measurements were made in the FT-ICR cell.
Table S3’. Product ions detected only in the linear ion trap following MS3 CID of the triply protonated,
[M + 2H]3+●, species of angiotensin I, DRVYIHPFHL.
18
Table S4. Product ions observed following MS3 CID of the singly protonated, M+●, species of [Ile7]angiotensin III, RVYIHPI-OH. The numbers in parenthesis indicate the product ions abundances in the
linear ion trap. All other measurements were made in the FT-ICR cell.
Table S4’. Product ions detected only in the linear ion trap following MS3 CID of the singly protonated,
M+●, species of [Ile7]-angiotensin III, RVYIHPI-OH.
19
Table S5. Product ions observed following MS3 CID of the doubly protonated, [M + H]2+●, species of
[Ile7]-angiotensin III, RVYIHPI-OH. The numbers in parenthesis indicate the product ions abundances in
the linear ion trap. All other measurements were made in the FT-ICR cell.
Table S5’. Product ions detected only in the linear ion trap following MS3 CID of the doubly protonated,
[M + H]2+●, species of [Ile7]-angiotensin III, RVYIHPI-OH.
20
Table S6. Product ions observed following MS3 CID of the singly protonated, M+●, species of ACTH,
SYSMEHFRWG-OH. The numbers in parenthesis indicate the product ions abundances in the linear ion
trap. All other measurements were made in the FT-ICR cell.
21
Table S7. Product ions observed following MS3 CID of the doubly protonated, [M + H]2+●, species of
ACTH, SYSMEHFRWG-OH. The numbers in parenthesis indicate the product ions abundances in the
linear ion trap. All other measurements were made in the FT-ICR cell.
Table S7’. Product ions detected only in the linear ion trap following MS3 CID of the doubly protonated,
[M + H]2+●, species of ACTH, SYSMEHFRWG-OH.
22
Table S8. Product ions observed following MS3 CID of the doubly protonated, [M + H]2+●, species of
neurotensin, pELYENKPRRPYIL-OH. The numbers in parenthesis indicate the product ions abundances
in the linear ion trap. All other measurements were made in the FT-ICR cell.
Table S8’. Product ions detected only in the linear ion trap following MS3 CID of the doubly protonated,
[M + H]2+●, species of neurotensin, pELYENKPRRPYIL-OH
23
Table S9. Product ions observed following MS3 CID of the triply protonated, [M + 2H]3+●, species of
neurotensin, pELYENKPRRPYIL-OH. The numbers in parenthesis indicate the product ions abundances
in the linear ion trap. All other measurements were made in the FT-ICR cell.
24
Table S9’. Product ions detected only in the linear ion trap following MS3 CID of the triply protonated,
[M + 2H]3+●, species of neurotensin, pELYENKPRRPYIL-OH
Table S10. Product ions observed following MS3 IRMPD of the singly protonated, M+●, species of
angiotensin I, DRVYIHPFHL.
25
Table S11. Product ions observed following MS3 IRMPD of the doubly protonated, [M + H]2+●, species
of angiotensin I, DRVYIHPFHL.
26
Table S12. Product ions observed following MS3 IRMPD of the triply protonated, [M + 2H]3+●, species
of angiotensin I, DRVYIHPFHL.
27
Table S13. Product ions observed following MS3 IRMPD of the singly protonated, M+●, species of [Ile7]angiotensin III, RVYIHPI-OH.
Table S14. Product ions observed following MS3 IRMPD of the doubly protonated, [M + H]2+●, species
of [Ile7]-angiotensin III, RVYIHPI-OH.
28
Table S15. Product ions observed following MS3 IRMPD of the singly protonated, M+●, species of
ACTH, SYSMEHFRWG-OH.
29
Table S16. Product ions observed following MS3 IRMPD of the doubly protonated, [M + H]2+●, species
of ACTH, SYSMEHFRWG-OH.
30
Table S17. Product ions observed following MS3 IRMPD of the doubly protonated, [M + H]2+●, species
of neurotensin, pELYENKPRRPYIL-OH.
Table S17 continues on next page
31
Table S17 – continued.
32
Table S18. Product ions observed following MS3 IRMPD of the triply protonated, [M + 2H]3+●, species
of neurotensin, pELYENKPRRPYIL-OH.
Table S18 continues on next page
33
Table S18 - continued.
34
Table S19. Product ions observed following MS3 EID of the singly protonated, M+●, species of
angiotensin I, DRVYIHPFHL.
Table S20. Product ions observed following MS3 EID of the doubly protonated, [M + H]2+●, species of
angiotensin I, DRVYIHPFHL.
35
Table S21. Product ions observed following MS3 EID of the triply protonated, [M + 2H]3+●, species of
angiotensin I, DRVYIHPFHL.
36
Table S22. Product ions observed following MS3 EID of the singly protonated, M +●, species of [Ile7]angiotensin III, RVYIHPI-OH.
37
Table S23. Product ions observed following MS3 EID of the doubly protonated, [M + H]2+●, species of
[Ile7]-angiotensin III, RVYIHPI.
38
Table S24. Product ions observed following MS3 EID of the singly protonated, M+●, species of ACTH,
SYSMEHFRWG-OH.
39
Table S25. Product ions observed following MS3 EID of the doubly protonated, [M + H]2+●, species of
ACTH, SYSMEHFRWG-OH.
40
Table S26. Product ions observed following MS3 EID of the doubly protonated, [M + H]2+●, species of
neurotensin, pELYENKPRRPYIL-OH.
Table S26 continues on next page
41
Table S26 – continued.
42
Table S27. Product ions observed following MS3 EID of the triply protonated, [M + 2H]3+●, species of
neurotensin, pELYENKPRRPYIL-OH.
Table S27 continues on next page
43
Table S27 – continued.
44
Table S28. Observed amino acid side chain losses from the radical precursor ions of angiotensin I,
DRVYIHPFHL, in MS3 CID per individual amino acid and for each charge state examined.
‡
This side chain loss was only observed from the product ions.
Table S29. Observed amino acid side chain losses from the radical precursor ions of [Ile 7]-angiotensin
III, RVYIHPI, in MS3 CID per individual amino acid and for each charge state examined.
45
Table S30. Observed amino acid side chain losses from the radical precursor ions of ACTH,
SYSMEHFRWG-OH, in MS3 CID per individual amino acid and for each charge state examined.
Table S31. Observed amino acid side chain losses from the radical precursor ions of neurotensin,
pELYENKPRRPYIL-OH, in MS3 CID per individual amino acid and for each charge state examined.
46
Table S32. Observed amino acid side chain losses from the radical precursor ions of angiotensin I,
DRVYIHPFHL, in MS3 IRMPD per individual amino acid and for each charge state examined.
‡
This side chain loss was only observed from the product ions.
Table S33. Observed amino acid side chain losses from the radical precursor ions of [Ile 7]-angiotensin
III, RVYIHPI, in MS3 IRMPD per individual amino acid and for each charge state examined.
47
Table S34. Observed amino acid side chain losses from the radical precursor ions of ACTH,
SYSMEHFRWG-OH, in MS3 IRMPD per individual amino acid and for each charge state examined.
Table S35. Observed amino acid side chain losses from the radical precursor ions of neurotensin,
pELYENKPRRPYIL-OH, in MS3 IRMPD per individual amino acid and for each charge state examined.
48
Table S36. Observed amino acid side chain losses from the radical precursor ions of angiotensin I,
DRVYIHPFHL, in MS3 EID per individual amino acid and for each charge state examined. No side chain
losses were observed from the singly protonated radical cations.
Table S37. Observed amino acid side chain losses from the radical precursor ions of [Ile7]-angiotensin
III, RVYIHPI, in MS3 EID per individual amino acid and for each charge state examined.
49
Table S38. Observed amino acid side chain losses from the radical precursor ions of ACTH,
SYSMEHFRWG-OH, in MS3 EID per individual amino acid and for each charge state examined.
Table S39. Observed amino acid side chain losses from the radical precursor ions of neurotensin,
pELYENKPRRPYIL-OH, in MS3 EID per individual amino acid and for each charge state examined.
50