Page S1
Supplementary Material for:
Cyclization Reaction of Peptide Fragment Ions during
Multistage
Collisionally
Activated
Decomposition:
An
Inducement to Lose Internal Amino-Acid Residues
Chenxi Jia, Wei Qi*, Zhimin He
Chemical Engineering Research Center, School of Chemical Engineering and Technology,
Tianjin University, Tianjin, 300072, People’s Republic of China.
Address reprint requests to Dr. Wei Qi
Chemical Engineering Research Center, School of Chemical Engineering and Technology,
Tianjin University, Tianjin, 300072, People’s Republic of China.
Phone: +86-22-27407799
Fax
: +86-22-27404757
E-mail: [email protected]
Supplementary Material for CYCLIZATION REACTION OF FRAGMENT IONS
Page S2
b6
100
630.7
A761.9 [PFNSLAI+H]
+
b5
Relative Abundance
559.6
50
0
b5
a4
541.6
-H 2 O
*
0
b6
b6
612.7
613.7
743.9
y5
b3
359.4
-NH3
427.6
386.5
b6-NH3
446.5
517.6 531.6
400
726.9
595.7
648.7
0
300
+
761.9
-H 2 O
*
b4
z4
[M+H]
500
600
700
m/z
b5
Relative Abundance
100
558.7
B787.0 [PLIFSPI+H]
+
50
b5-H2O
y4
y3
a4
316.4
a5 540.7
443.6
b4
b3
b2
471.6
376.5
211.3
411.6
463.6
768.9
b6
y5
445.5
324.4
-H 2 O
530.7
[M+H]
655.8
+
787.0
576.7
0
200
300
400
500
600
700
800
m/z
Figure S1. CAD spectra (MS2) of the protonated peptides (A) PFNSLAI and (B) PLIFSPI. Similarly, no
anomalous ions with internal residue losses are observed obviously in the MS2 spectra of other
protonated peptides in this investigation.
Supplementary Material for CYCLIZATION REACTION OF FRAGMENT IONS
Page S3
b5
Relative Abundance
100
558.7
A787.0 [IFSPIPL+H]
-H 2 O
+
769.0
b5-H2O
540.7
a5
530.7
50
y5
526.7
y4
439.5
[M+H]
+
787.0
b3
b4
348.4
y5-H2O
445.5
b6
508.7
655.8
0
300
400
500
600
700
800
m/z
-H 2 O
100
811.0
B829.0 [CH CO-IFSPIPL+H]
E
b5
+
600.7
3
E
b4 -CH2CO
655.8
Relative Abundance
b5-H2O
y4
439.6
E
b3
582.7
E
b4 -H2O
460.5
y6
390.4
50
y3
342.5
300
673.8
E
b4
×5
487.5
400
500
600
700
[M+H]
E
E
b5 -H2O
E
b5
b4 -CH2CO
600.7
655.8
582.7
+
829.0
y6
673.8
0
300
400
500
600
700
800
m/z
Figure S2. CAD spectra (MS2) of (A) the protonated peptide IFSPIPL and (B) the protonated N-acetylated
peptide CH3CO-IFSPIPL.
Supplementary Material for CYCLIZATION REACTION OF FRAGMENT IONS
Page S4
a5-S
×5
100
443.7
A787.0 [IFSPIPL+H]
558.7 IFSPI
Relative Abundance
471.6 PIIF
+
+
b5
+
b5-FS
324.4
b5-S
[b5-S]-I
358.5
50
{[b5-S]-P}
[b5-S]-FP
374.5
227.3
b5-S
b5-IFS
471.6
211.3
a5-IFS
{[b5-S]-PI}
183.3
[b5-S]0
329.6
261.3
453.6
0
150
200
250
300
350
400
450
500
m/z
a5-F
100
383.7
B787.0 [IFSPIPL+H]
558.7 IFSPI
+
Relative Abundance
411.5 SPII
+
+
b5
[b5-F]0
b5-F
393.5
b5-IF
298.4
50
[b5-F]-P
[b5-F]-IS
[b5-IF]
{[b5-F]-S}
0
b5-F
280.4
411.5
[b5-F]-PI
{[b5-F]-SP}
[a5-F]-IS
[a5-F]
*
366.5
211.3
183.3
201.3
326.5
324.4
227.3
314.4
0
100
150
200
250
300
m/z
Supplementary Material for CYCLIZATION REACTION OF FRAGMENT IONS
350
400
Page S5
0
a5
100
512.7
C787.0 [IFSPIPL+H]
558.7 IFSPI
+
+
b5
+
0
Relative Abundance
540.7 IFSPI -H2O b5
50
0
b5-F-NH3
0
0
b4
{b5-IF}
427.5
280.6
0
0
376.4
a5-IF-H2O
b5-NH3
0
523.7
b5-F
0
393.5
0
b5-IIF
0
a5-IF
b3
252.6
348.4
308.4
167.5
195.3 211.3
314.5
b5
540.7
495.7
0
a4
399.7
263.4
234.5
0
a5-NH3
b4
445.5
0
150
200
250
300
350
400
450
500
550
m/z
Figure S3.
CAD spectra (MS4) of the (A) b5–S, (B) b5–F and (C) b50 ions. These three parent ions arise from
CAD spectrum (MS3) of the b6 ion from peptide IFSPIPL (Figure 3A).
Supplementary Material for CYCLIZATION REACTION OF FRAGMENT IONS
Page S6
0
b5
×7
100
787.0 [PLIFSPI+H]
+
558.7 PLIFS
540.7
+
b5-I
b5-L
8000 ms
b5
445.5
Relative Abundance
8000 ms
*
a5-H2O
b5-LI
b5-IF
298.6
b4+H2O-LI
b4+H2O-I
b3
376.5
-H2O
324.4
b5-F
{b5-PL}
b5-F
b5-IF
393.5
280.6
513.7
427.5
332.5
342.4
211.3
F
530.7
*
a5
471.6
0
0
0
b5-NH3
a5
b4
411.5
348.5
263.3
b2
b4+H2O
b3+H2O
50
495.7
489.6
523.7
b5
558.7
120.1
0
100
200
300
400
500
m/z
Figure S4. CAD spectra (MS3) of the b5 ion from peptide PLIFSPI. The spectra were acquired with an
activation time of 8000 ms at each stage of CAD.
Supplementary Material for CYCLIZATION REACTION OF FRAGMENT IONS
Page S7
Intensity Ratio
4
(b5-L&I)/b4
(b5-F)/b4
(b5-L&I)/(b5-F)
3
2
b3/b4
1
0
2000
4000
6000
8000
Activation Time (ms)
Figure S5. Effect of the activation time on the fragment-ion intensity relationship of the b5 ion from peptide
PLIFSPI. The same collision energy was used in these CAD experiments; the same activation
time was used at the two stage of CAD. b5–L&I represents the ion at m/z 445.5 in Figure 1B.
cyclo(PFNSLA)
Supplementary Material for CYCLIZATION REACTION OF FRAGMENT IONS
Page S8
n
bnPA
bnFP
bnNF
bnSN
bnLS
bnAL
6
A
630.7
P
630.7
F
630.7
N
630.7
S
630.7
L
630.7
5
L
559.6
A
533.6
P
483.5
F
516.6
N
543.6
S
517.6
4
S
446.5
L
462.5
A
386.4
P
369.4
F
429.5
N
430.5
3
N
359.4
S
349.4
L
315.3
A
272.3
P
282.4
F
316.4
2
F
245.3
N
262.3
S
202.2
L
201.2
A
185.2
P
169.2
1
P
98.1
F
148.2
N
115.1
S
88.1
L
114.2
A
72.1
×16
100
-NH3
×8
630.7 [cyco(PFNSLA)+H]
-H2O
+
613.7
612.7
b3PA
Relative Abundance
359.4
b4LS
429.6
b4AL
0
a5SN
430.5
470.6
b4SN
0
b5SN-NH3
369.4
50
b3PA+H2O
377.4
a5SN
b4PA
488.5
b5PA
446.5
b3NF
559.6
0
-CO
b5SN
b4NF
0
-NH3
b3NF
b2NF
b2PA
a3PA
245.3
217.3
498.6
331.4
a2PA
202.3
595.6
386.5
b5SN
516.6
315.5
481.5
297.5
b5LS
[M+H]
543.8
602.7
+
630.7
0
200
300
400
500
600
m/z
Figure S6. CAD spectrum (MS2) of the protonated cyclic peptide cyclo(PFNSLA). This cyclic peptide has
been sequenced using multistep CAD in our previous work (Ref. [26]). The above table lists the
calculated m/z values of b ions of the cyclic peptide, which are used to designate the fragment
ions in the CAD spectrum. The nomenclature for labeling fragment ions of cyclic peptides is
proposed by Ngoka and Gross (J. Am. Soc. Mass Spectrom. 1999, 10, 360-363). Ions are tagged
with a four-part descriptor with the general formula xnJZ, where “x” is the designation for the ion
and n is the number of amino acid residues in the ion. J and Z are the one-letter codes for the two
amino acid residues connecting the backbone amide bond, J-Z, that was broken to form the linear
ion. J is the N-terminal amino acid residue and Z is the C-terminal amino acid residue.
Supplementary Material for CYCLIZATION REACTION OF FRAGMENT IONS
Page S9
*
a5-SP
100
329.5
A787.0 [IFSPIPL+H]
+
+
530.7 IFSPI -CO a5
*
Relative Abundance
416.5 a5-P
50
[a*5-P]-I
*
a5-SP-CO
[a*5-SP-CO]-F
*
a5-P
416.5
303.4
*
*
154.5
[a5-SP-CO]-I
a5-P-CO
301.4
387.5
188.5
0
100
150
200
250
300
350
400
m/z
*
a5-IF
100
253.5
B787.0 [IFSPIPL+H]
+
+
530.7 IFSPI -CO a5
*
*
a5-F
Relative Abundance
366.6 a5-F
-H2O
366.6
348.6
50
*
a5-F-CO
338.4
-H2O
320.2
227.7
0
100
150
200
250
300
350
m/z
Figure S7. CAD spectra (MS4) of the (A) a5*–P and (B) a5*–F ions. These two parent ions arise from CAD
spectrum (MS3) of the a5 ion from peptide IFSPIPL (Figure 6).
Supplementary Material for CYCLIZATION REACTION OF FRAGMENT IONS
Page S10
E
b4
100
487.5
829.0 [CH3CO-IFSPIPL+H]
+
+
E
Relative Abundance
572.7 CH3CO-IFSPI -CO a5
E
b3
50
390.5
E
a5
572.7
E
a4 -CH2CO
183.6
417.5
E
a5 -H2O
E
b4 -H2O
554.7
469.7
270.7
0
100
200
300
400
500
600
m/z
Figure S8.
CAD spectrum (MS3) of the a5E ion from N-acetylated peptide CH3CO-IFSPIPL.
Supplementary Material for CYCLIZATION REACTION OF FRAGMENT IONS