Functional Microgelsassisted Tryptic Digestionand Quantification of Cytochrome Cthroughinternal

Supporting Information

Functional MicrogelsAssisted Tryptic Digestionand Quantification of Cytochrome cthroughInternal Standard Mass Spectrometry

Li-Yi Chen,1Wei-Cheng Wu,2,3and Huan-Tsung Chang*,1

1Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan; 2Department of Engineering and System Science, National Tsing Hua University, Hsinchu 30013, Taiwan; 3Nano Science and Technology Program, Taiwan International Graduate Program, Academia Sinica, Taipei 11529, Taiwan

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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.

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.

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.

Fig.S4Plotsof the ratios of (a) the sum of two MS signals tointernal standard signal ((I779.5+I1168.6)/I1067.6)and (b) the sum of six MS signals to internal standardsignal ((I779.5+I817.5+I907.6+I1168.6+I1296.6+I1478.8)/I1067.6)against the concentration of Cytc over the range 25–200nM. 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

Fig. S5MS spectra of tryptic digests of potential protein interferences through SALDI-MS. Asterisks denotebackground MS signals from Au NPs. Conditions are the same as that in Fig. 2a. The concentration of each protein is 500nM.

Fig. S6Cell 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-1carbon dots for 24 h. Cell viability was analyzed by Alamar Blue method. The percentages of cell viability are shown as a relative value to control (untreated). The value represent mean ± standard deviation (n = 3).