Supporting Information
Functional Microgels Assisted Tryptic Digestion and
Quantification of Cytochrome c through Internal
Standard Mass Spectrometry
Li-Yi Chen,1 Wei-Cheng Wu, 2,3and Huan-Tsung Chang*,1
1
Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan; 2Department
of Engineering and System Science, National Tsing Hua University, Hsinchu 30013, Taiwan;
3
Nano Science and Technology Program, Taiwan International Graduate Program, Academia
Sinica, Taipei 11529, Taiwan
Address reprint requests to
Professor Huan-Tsung Chang, National Taiwan University, Department of Chemistry
1, Section 4, Roosevelt Road,Taipei 106, Taiwan
Tel. and fax: 011-886-2-33661171
E-mail: changht@ntu.edu.tw
1
Fig. S1 (a) Zeta potentials of MGs (1 mg mL-1), Au NPs/MGs (2X) and TR/Au NPs/MGs
(0.15 mg mL-1 trypsin/2X Au NPs/MGs) in phosphate solutions (10 mM) at various pH
values. (b) Time-dependent immobilization efficiency of trypsin (0.25 mg mL-1) in the Au
NPs/MGs (2X) in Tris-HCl solution (10 mM, pH 7.3).
Error bars represent standard
deviations from three replicate measurements.
2
Fig. S2 Effect of microwave irradiation time on (a) tryptic digestion efficiency and (b)
specificity of correct over missed digestion of Cyt c (100 nM).
TR/Au NPs/MGs in
NH4HCO3 solution (10 mM, pH 8.3) were used for digestion. (a) MS signal ratio
(I1168.6/I1067.6); (b): Relative ratio of the specific cleavage peptide (I1168.6) over that of missed
cleavage peptide (I1296.6), assuming that their sum is equal to 100% in each run. I1067.6
represents the signal intensity of internal standard.
3
Fig. S3 Reusability of TR/Au NPs/MGs. Each digestion was performed in NH4HCO3
solution (10 mM, pH 8.3) containing Cyt c (50 nM) under microwave irradiation for 15 s.
Error bars represent standard deviations from three replicate measurements.
4
Fig. S4 Plots of the ratios of (a) the sum of two MS signals to internal standard signal ((I779.5
+ I1168.6)/I1067.6) and (b) the sum of six MS signals to internal standard signal ((I779.5 + I817.5 +
I907.6 + I1168.6 + I1296.6 + I1478.8)/I1067.6) against the concentration of Cyt c over the range 25–200
nM. These MS signals were selected from tryptic digest of Cyt c at m/z value of 779.5,
817.5, 907.6, 1168.6, 1296.6, 1478.8 and internal standard (GR-10) at m/z value of 1067.6.
Error bars represent standard deviations from three replicate measurements
5
Fig. S5 MS spectra of tryptic digests of potential protein interferences through SALDI-MS.
Asterisks denote background MS signals from Au NPs.
Conditions are the same as that in
Fig. 2a. The concentration of each protein is 500 nM.
6
Fig. S6 Cell viability of HeLa cells after being treated with (a) etoposide and (b) carbon dots.
HeLa cells (4  103 cells per well) were seeded and grown for 16 h.
The cells were then
incubated with (a) 5–100 M etoposide and (b) 0.19–1.5 mg mL-1 carbon dots for 24 h.
Cell viability was analyzed by Alamar Blue method.
shown as a relative value to control (untreated).
The percentages of cell viability are
The value represent mean ± standard
deviation (n = 3).
7