SUPPORTING INFORMATION
Table S1. List of tryptic peptides generated from the modified protein ubiquitin (1:250)
in the ESI-FTICR-MS spectrum
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Residues
Expected m/z
Observed m/z
Sequence
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28-42
1806.96 (+1)
903.97 (+2)/
AK*IQDK*EGIPPDQQR
602.99 (+3)
43- 54
1388.76 (+1)
694.88 (+2)
LIFAGK*QLEDGR
55- 72
2172.18 (+1)
1086.59 (+2)/
TLSDYNIQK*ESTLHLVLR
724.72 (+3)
* Guanidinated
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Table S2. List of tryptic peptides (m/z) generated from the modified protein ubiquitin
(1:100) in the ESI-FTICR-MS spectrum.
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Peptides with no missed cleavage
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Residues
Expected m/z
Observed m/z
Sequence
1- 06
807.45 (+1)
807.45 (+1)
MQIFVK*
7- 11
561.34 (+1)
561.34 (+1)
TLTGK*
43- 48
690.43 (+1)
690.43 (+1)
LIFAGK*
49- 54
717.35 (+1)
717.35 (+1)
QLEDGR
64- 72
1067.62 (+1)
1067.59 (+1)/
ESTLHLVLR
534.31 (+2)
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Peptide with 1, 2 missed cleavage
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28-42
1806.96 (+1)
903.97 (+2)/
AK*IQDK*EGIPPDQQR
602.99 (+3)
43- 54
1388.76 (+1)
694.88 (+2)
LIFAGK*QLEDGR
55- 72
2172.18 (+1)
1086.59 (+2)/
TLSDYNIQK*ESTLHLVLR
724.72 (+3)
55- 76
2555.4052
852.46 (+3)
TLSDYNIQK*ESTLHLVLRLRGG
* Guanidinated
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Table 3. List of tryptic peptides generated from the modified ubiquitin (1:25) in ESIFTICR-MS spectrum
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Peptides with no missed cleavage
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Residues
Theoretical m/z Observed m/z
Sequence
1- 06
807.45 (+1)
807.45 (+1)
MQIFVK*
7- 11
561.34 (+1)
561.34 (+1)
TLTGK*
12- 27
1829.95 (+1)
915.48 (+2)
TITLEVEPSDTIENVK*
34- 42
1039.52 (+1)
1039.52 (+1)
EGIPPDQQR
43- 48
690.43 (+1)
690.43 (+1)
LIFAGK*
49- 54
717.35 (+1)
717.35 (+1)
QLEDGR
55- 63
1123.57 (+1)
562.29 (+2)
TLSDYNIQK*
64- 72
1067.62 (+1)
1067.60 (+1)
ESTLHLVLR
534.31 (+2)
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Peptide with 1, 2 and 3 missed cleavage
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12- 29
2071.10 (+1) 1036.06 (+2)
TITLEVEPSDTIENVK*AK*
30- 42
1565.80 (+1) 783.41 (+2)
IQDK*EGIPPDQQR
43- 54
1388.76 (+1) 694.88 (+2)
LIFAGKQLEDGR
55- 72
2172.18 (+1) 1086.59 (+2)/
TLSDYNIQK*ESTLHLVLR
724.72 (+3)
7- 29
2613.42 (+1)
871.81 (+2)
TLTGK*TITLEVEPSDTIENVK*AK*
28- 42
1806.96 (+1) 602.99 (+3)
AK*IQDK*EGIPPDQQR
30- 48
2237.21 (+1)
746.41 (+3)
IQDK*EGIPPDQQRLIFAGK*
* Guanidinated
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Figure S1. ESI-FTICR-MS/MS spectra of homoarginine and arginine-terminated tryptic
peptides from the modified protein ubiquitin. (A) MS/MS spectrum of a homoarginineterminated peptide, m/z 807.45 (residue 1-6). (B) ESI-FTICR-MS/MS spectrum of the
most intense peak (m/z 694.89) observed in the mass spectra of Figs. 1(B) and 1(C). This
peptide has one missed cleavage at the homoarginine site. The homoarginine-terminated
peptide was also observed at 1:100 and 1:25 protease/protein ratios (accurate m/z 690.43)
(see Figs. 2(B) and 2(C)).
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Figure S2. MALDI-TOF-MS spectra of the modified ubiquitin at different time points at
1:50 protease/protein ratio. The homoarginine-terminated peptides m/z 807.48 appeared
as low intensity peaks in the spectra in 30 min after digestion. The intensity of this
peptide was increased with digestion time.
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Figure S3. ESI-FTICR-MS spectra of modified and unmodified ubiquitin at 0.5 h at 1:50
protease/protein ratio. (A) ESI-FTICR-MS spectrum of the digest of modified ubiquitin
in 0.5 h. (B) ESI-FTICR-MS spectrum of the tryptic digest of unmodified ubiquitin in 0.5
h. K: lysine-terminated peptide, HR: homoarginine-terminated peptide, R: arginineterminated peptide.
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Figure S4. MALDI-TOF-MS spectrum of the tryptic digest of homoarginine-modified
protein β-casein. The calculated homoarginine cleavage sites are m/z 822.53, 957.50,
998.52 and 1097.43.
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Figure S5. MALDI-TOF mass spectra of the selective enrichment of blocked peptides
from an N-terminus blocked and unblocked ubiquitin protein digest. (A) MALDI-TOF
mass spectrum of the mixture of N-termini blocked and unblocked ubiquitin digest. Cterminal lysines in the peptides were converted into homoarginine and methionine
residues were oxidized with performic acid (guanidinated) and N-termini were blocked
by acetylation (acetylated). (B) MALDI-TOF mass spectrum of the supernatant after
solid-phase capture. All acetylated peptides were enriched by this reverse solid-phase
purification strategy.
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1102.09
x10 4
N-term acetylated
Bradykinin (Ac-RPPGFSPFR)
4
946.09
Intens . [ a.u .]
5
3
N-term acetylated bradykinin fragment (Ac-RPPGFSPF)
2
1
0
750
1000
1250
1500
1750
2000
2250
m/z
Figure S6. MALDI-TOF mass spectrum of acetylated bradykinin alone. Spectrum
showed the presence of an acetylated bradykinin after loss of the C-terminal arginine
residue.
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S-peptide from RNase S
protein (K*ETAAAK*FER)
100
638.84
2+
74
Ac-M(*O2)QIFVK*(1+)
Ubiquitin N-term
Ac-RPPGFSPFR (2+)
881.46
Spiked
Ac-RPPGFSPFR (1+)
1102.57
1276.68 (1+)
37
426.24
0
200
Ac-RPPGFSPF
946.48
551.79
650
m/z 1100
1550
2000
Figure S7. ESI-FTICR-MS mass spectrum of the N-terminal peptides enriched from the
RNaseS protein and ubiquitin by this solid-phase purification strategy. Spiked N-terminal
acetylated bradykinin and the acetylated bradykinin fragment were also observed in the
spectrum. See Fig. 4 for MALDI-TOF mass spectrum. K*: homoarginine (HR)-modified
lysine.
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Figure S8. MALDI-IT-TOF mass spectrum of unmodified ubiquitin and modified
ubiquitin under 1:100 and 1:50 protease/protein ratio at 12 h digestion period. See Fig. 2
for comparison.
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