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Table 1 SM. MASCOT score, protein sequence coverage, and number of identified peptides obtained after PFM analysis of single protein digests (3 h digestion at 37°C and 5 min ultrasonic digestion at 50% ultrasonic amplitude). Three replicates were done for each experimental condition.

Table 2SM. Proteins identified in each protein mixture by MS/MS after 5 min of ultrasonication with 50% ultrasonic amplitude. Three replicates were done for each experimental condition.

Fig. 1SM. MALDI spectra of 50 ng of protein digested on indium tin oxide (ITO)-coated glass slide (0.5 μL of solution containing 2.5 ng/μL of trypsin in 25 mM ammonium bicarbonate). Panels (A, E, I, and M–α-lactalbumin; B, F, J, N–lysozyme; C, G, K, and O–carbonic anhydrase; D, H, L, and P–serum albumin). Panels (A–D) proteins were digested in 5 min with sonoreactor (2.5 + 2.5 min) at 50% of sonication amplitude. Panels (E–H) proteins were digested in 5 min without ultrasonication. Panels (I–L) proteins were digested in humidified chamber for 3 h at 37°C. Panels (M–P) proteins were digested in humidified chamber for 5 min at 37°C.

Fig. 2SM. MALDI spectra of 50 ng of protein digested on ITO-coated glass slide (0.5 μL of solution containing 2.5 ng/μL of trypsin in 25 mM ammonium bicarbonate). Panel A–α-lactalbumin; B–carbonic anhydrase; C–Lysozyme; D–phosphorylase b. Proteins were digested 5 min without the use of ultrasonication.

Fig. 3SM. MALDI spectra of 50 ng of protein spotted on ITO-coated glass slide (without the aid of trypsin) and sonicated for 5 min with sonoreactor (2.5 + 2.5 min). Panels A–α-lactalbumin; B–carbonic anhydrase; C–Lysozyme; D–phosphorylase b.

Fig. 4SM. MALDI spectra of 50 ng of protein spotted on ITO-coated glass slide. Proteins were on-target reduced, on-target alkylated, and digested for 5 min (2.5 + 2.5 min) with sonoreactor at 50% of sonication amplitude. Panel A–α-lactalbumin and B–carbonic anhydrase.

Fig. 5SM. MALDI spectra of mixtures of protein digested on ITO-coated glass slide (0.5 μL of solution containing 2.5 ng/μL of trypsin in 25 mM ammonium bicarbonate). Panels A and D–α-lactalbumin (8 ng), carbonic anhydrase (15 ng), lysozyme (8 ng), and phosphorylase b (50 ng); B and E–α-lactalbumin (8 ng), carbonic anhydrase (7.5 ng), lysozyme (8 ng), and phosphorylase b (25 ng); C and F α-lactalbumin (4 ng), carbonic anhydrase (15 ng), lysozyme (4 ng), and phosphorylase b (50 ng). Panels A, B, and C protein mixtures were digested for 5 min (2.5 + 2.5 min) with sonoreactor at 50% of sonication amplitude. Panels D, E, and F were digested in humidified chamber for 3 h at 37°C.

Fig. 6SM. MALDI spectra of mixtures of protein digested on ITO-coated glass slide (0.5 μL of solution containing 2.5 ng/μL of trypsin in 25 mM ammonium bicarbonate). Mix I–α-lactalbumin (8 ng), carbonic anhydrase (15 ng), lysozyme (8 ng), and serum albumin (25 ng); Mix II–α-lactalbumin (8 ng), carbonic anhydrase (7.5 ng), lysozyme (8 ng), and serum albumin (12.5 ng); Mix III–α-lactalbumin (4 ng), carbonic anhydrase (15 ng), lysozyme (4 ng), and serum albumin (25 ng). Panels (A–C) samples were digested in 5 min with sonoreactor (2.5 + 2.5 min) at 50% of sonication amplitude. Panels (D–F) proteins were digested in humidified chamber for 3 h at 37°C. Panels (G–I) proteins were digested in 5 min without ultrasonication. Panels (J–L) proteins were digested in humidified chamber for 5 min at 37°C.

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