Electronic Supplementary Information

Increased Disulfide Peptide Sequence Coverage via “Cleavage

ON/OFF” Switch during Nanoelectrospray

Gongyu Li, Yue Yin, and Guangming Huang*

Detailed Experimental Settings.

Table S1. Partial oxygen addition fragment ions observed in InESI-CID-MS analysis and sequence

coverage for selectin binding peptide and somatostatin.

Scheme S1. Possible oxidative cleavage reaction pathway for the cleavage of DSB via radical generation

in InESI.

Fig. S1. MS spectrum of the oxidative cleavage products from selectin binding peptide by InESI.

Fig. S2. MS3 spectra of the oxidative cleavage products from selectin binding peptide by InESI.

Fig. S3. The increase trend of GSH, a pair-product of GSO• from DSB oxidative cleavage, with the

increase of amplitude of the pulsed high voltage (1 kHz). Corona discharge and DSB cleavage was

observed with a similar increase trend with adjusting of the amplitude of pulsed high voltage.

Fig. S4. Dependence of DSB dissociation on the frequency of pulsed high voltage (6 kV). In all these

experiments, NH4OAc (5 mM) was added into the spray solution.

Fig. S5. a) Comparison of oxidative cleavage reaction products from GSSG via InESI with different

spray voltages and different electrolytes. Dependence of DSB dissociation on low concentration volatile

electrolyte and amplitude of pulsed high voltage, as illustrated by b) GSO• and c) GSH. Different

concentrations of NH4OAc were indicated.

Fig. S6. a) Sonic spray ionization MS spectrum of GSSG in the presence of various concentrations of

NH4OAc. b) Magnification of a) for mass range between m/z 290 and 420. No oxidative cleavage

reaction cleavage products (GSH, m/z 308 or GSO•, m/z 323) were observed in sonic spray ionization for

the absence of corona discharge.

Electronic Supplementary Material (ESI) for RSC Advances.This journal is © The Royal Society of Chemistry 2014

Fig. S7. Tandem MS spectrum (low m/z range) of [M+5H]5+ for human insulin containing 10 mM

NH4OAc derived from “OFF” mode. Sequence information beyond the disulfide bonds were obtained.

Fig. S8. Reaction MS1 spectrum of insulin and isotopic distributions of OBb7+ and HBy20

2+ in InESI-CID-

MS spectrum of HO• additive precursor ion ([M + 5H + OH]5•+, m/z 1166) of human insulin. It should be

noted that the m/z 1166.0 in MS spectrum of insulin would be assumed to be NH4+ adduction product.

We excluded this possibility to some extent via CID tandem mass spectrometry and finally affirmed the

possibility of radical additive ion.

Detailed Experimental Settings.

For nanoESI:

The size of spray emitter was kept 5-10 µm. The emitters were pulled from borosilicate glass

capillaries (1.2 mm o.d., 0.9 mm i.d.) by using a P-2000 laser-based micropipette puller (Sutter

Instruments, Novato, CA, USA). Spray voltage for nanoESI was optimized and specified for each

experiment but was in the range of 1.0-2.0 kV dc. Tip to MS inlet was kept 5 mm for all nanoESI

operations. Spray solvent was also optimized and H2O was used as solvent if not specified.

For InESI:

For InESI, induced voltage was typically 4.0-12.0 kVp-p, 1 kHz, duty cycle 50%, pulsed high voltage.

This pulsed high voltage was originally generated from a 25 MHz function waveform generator

(DG1022U, RIGOL, Beijing, China) and was then amplified by a household stereo power amplifier. As a

result, 100 Vp-p pulsed voltage was obtained and followed a coil to further amplify the amplitude up to a

final value of 0-20 kVp-p. The high voltage was monitored with a digital oscilloscope (DS1052E, RIGOL,

Beijing, China) after attenuating (1: 1000) with a attenuation rod. For the consideration of security and

effectiveness of radical generation, 4-12 kVp-p (1 kHz) was selected in our experiments. Other operation

parameters for InESI were kept the same with nanoESI.

For MS:

All experiments were carried out with a LTQ-Velos Pro (Thermo Fisher Scientific, CA, USA) mass

spectrometer equipped with an linear ion trap. MS inlet capillary temperature was kept 275 °C. Positive

mode was operated for all cleavage identification experiments with S-lens value of 42%. CID energy was

optimized to obtain considerable fragment ions and 15% was finally used if not specified (Insulin, 27%).

Maximum injection time of ion trap was set as 10 ms for small peptides and 500 ms for insulin radical ion

fragmentation to obtain acceptable MS2 signal, and all are with 3 microscans. Isolation width was set as

1.0.

“Cleavage ON/OFF” experiment:

Cleavage ON/OFF tests were exemplified with GSSG (8 µM, 10 mM NH4OAc) and insulin (34.4 µM,

50 mM NH4OAc). For “Cleavage ON” mode was operated as follows, pulsed high voltage (1 kHz, 7 kV)

was applied on the ring electrode outside the tip for InESI operation and DC high voltage for

conventional nanoESI was meanwhile turned off. For “Cleavage OFF” mode, DC high voltage (1 kV)

was applied on the electrode inside the nanotip for nanoESI operation and pulsed high voltage for InESI

was meanwhile turned off. In order to avoid possible electric field interfere from induced voltage,

nanoESI electrode was withdrawed when operating “Cleavage ON” mode.

Table S1. Partial oxygen addition fragment ions observed in InESI-CID-MS analysis and sequence

coverage for selectin binding peptide and somatostatin.

Peptide Partial oxygen addition fragment ions observed, m/z Sequence CoverageOb4

+ Oy4+ Ob5

+ Oy5+ Oy6

+ Ob7+ Oy7

+ Ob8+selectin binding

peptide 474 492 587 605 718.5 786 847.5 942.5Oy7

+ Ob8+ Oy8

+ Ob9+ Oy9

+ Ob10+ Oy10

+ Ob11+

somatostatin888 970 1035 1098 1182 1199 1296 1346.6

Scheme S1. Possible oxidative cleavage reaction pathway for the cleavage of DSB via radical generation

in InESI.

Fig. S1. MS spectrum of the oxidative cleavage products from selectin binding peptide by InESI.

Fig. S2. MS3 spectra of the oxidative cleavage products from selectin binding peptide by InESI.

0

1000

2000

17000

ESL

Inte

nsityCorona discharge

4 6 8 10

0.4

0.8

1.2

GSH/GSSG

I GSH /

I GSSG

Pulsed HV / kV

DSB cleavage

Fig. S3. The increase trend of GSH, a pair-product of GSO• from DSB oxidative cleavage, with the

increase of amplitude of the pulsed high voltage (1 kHz). Corona discharge and DSB cleavage was

observed with a similar increase trend with adjusting of the amplitude of pulsed high voltage.

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.000.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

I 308 /

I (307

+613

) (/20

)

Pulsed frequency / kHz

I GSO. /

I GSSG

GSO./GSSG GSH/GSSG

Fig. S4. Dependence of DSB dissociation on the frequency of pulsed high voltage (6 kV). In all these

experiments, NH4OAc (5 mM) was added into the spray solution.

a)

b) c)

GSSG_6 kV

GSSG + 5 mM NH4OAc_6 kV

GSSG + 5 mM NH4OAc_12 kV

GSSG + 5 mM NH4HCO3_6 kV

307

356323340324 372

308

[ GSSG + 2H ] 2+

[ GSH + H ] +

[ GSO• + H ] +•

+ O + 2O + 3O

m/z310 350 400

4 6 8 10 120.0

0.4

0.8

1.2GSH/GSSG

I GSH /

I GSSG

Pulsed HV / kV

5 mM 10 mM 20 mM

4 6 8 10 120.000

0.015

0.030

0.045

0.060

0.075GSO./GSSG

Pulsed HV / kV

5 mM 10 mM 20 mM

I GSO. /

I GSSG

Fig. S5. a) Comparison of oxidative cleavage reaction products from GSSG via InESI with different

spray voltages and different electrolytes. Dependence of DSB dissociation on low concentration volatile

electrolyte and amplitude of pulsed high voltage, as illustrated by b) GSO• and c) GSH. Different

concentrations of NH4OAc were indicated.

GSSG+10 mM NH4OAc

GSSG+50 mM NH4OAc

GSSG+500 mM NH4OAc

GSSG

a)

b)

GSSG+10 mM NH4OAc

GSSG+50 mM NH4OAc

GSSG+500 mM NH4OAc

GSSG

300 310 320 330 340 350 360 370 380 390 400 410m/z

0

50

1000

50

1000

50

100

Rel

ativ

e A

bund

ance

0

50

100307.09

326.09318.09 337.17301.17 345.00 415.26352.25 388.25371.25 401.09379.25361.00307.09

337.25

301.17 371.34 391.34338.25317.09 326.09 352.25 415.26409.26365.17 385.34 402.26337.17

307.09

391.34352.25 371.25338.25326.09301.17 317.09 409.17365.25 385.17 403.01337.25

341.25 385.25297.17 307.09 371.34 391.25344.25 354.34315.09 365.17326.09 409.17401.26379.25

300 350 400 450 500 550 600 650m/z

0

50

1000

50

1000

50

100

Rel

ativ

e A

bund

ance

0

50

100307.09

613.18

635.18 651.18326.09536.17473.26 610.26432.26415.26345.00 388.25 520.34 564.42

613.18

307.09462.17 536.17337.25

610.26 635.18 651.18371.34 391.34 445.17326.09 520.42 564.42429.34 497.26 585.34613.18

536.17462.17337.25307.09 610.26473.34 635.18 651.18391.34371.25 445.17 550.26429.42 520.34 579.34613.26

635.18337.25 462.17 651.18536.17 561.42 605.43517.42429.34 476.34297.17 385.25371.34

Fig. S6. a) Sonic spray ionization MS spectrum of GSSG in the presence of various concentrations of

NH4OAc. b) Magnification of a) for mass range between m/z 290 and 420. No oxidative cleavage

reaction cleavage products (GSH, m/z 308 or GSO•, m/z 323) were observed in sonic spray ionization for

the absence of corona discharge.

Fig. S7. Tandem MS spectrum (low m/z range) of [M+5H]5+ for human insulin containing 10 mM

NH4OAc derived from “OFF” mode. Sequence information beyond the disulfide bonds were obtained.

Fig. S8. Reaction MS1 spectrum of insulin and isotopic distributions of OBb7+ and HBy20

2+ in InESI-CID-

MS spectrum of HO• additive precursor ion ([M + 5H + OH]5•+, m/z 1166) of human insulin. It should be

noted that the m/z 1166.0 in MS spectrum of insulin would be assumed to be NH4+ adduction product.

We excluded this possibility to some extent via CID tandem mass spectrometry and finally affirmed the

possibility of radical additive ion.