A ratiometric lectin microarray approach to analysis of the dynamic mammalian glycome

Piobello et al. 10.1073/pnas.0704954104.

Supporting Information

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Fig. 6. Single-color glycan analysis of Pro-5 samples labeled pre- and postlysis. Data shown are the average signal over 10 spots with standard deviations. Although labeling of the sample prelysis gives lower dye incorporation levels, the glycopatterns are the same for both samples. It should be noted that because of newly optimized protocols for lectin microarray printing, the previously borderline ECA lectin now gives a positive signal for Pro-5.

Fig. 7. GlcNAc inhibition of select Pro-5 and Lec8 signals. Arrays were preincubated with 200 mM GlcNAc before hybridization of Pro-5 or Lec8 samples. Data are shown as % inhibition of the signal, i.e., ((Average Signal from Control Array - Average Signal from GlcNAc-inhibited Array)/Average Signal from Control Array) ´ 100. Error was propagated from the standard deviations. Excellent to modest inhibition of the lectin signals were observed dependent on both the lectin and the cell line. Relative inhibition levels of WGA for Pro-5 vs. Lec8 may reflect the loss of tighter binding epitopes on Lec8 for this lectin.

Fig. 8. Single-color glycan analysis of Pro-5 and Lec8. Data shown are the average signal over 10 spots with standard deviations. Pro-5 CHO cells were characterized by strong signals for ConA, LcH, MAA, and WGA, moderate signals for AIA and SNA, and weak signals for DSA, GNA, and HPA. The positive signals for the single color Lec8 experiments overlapped with those of Pro-5 CHO with a few exceptions. For Lec8, HPA and GNA appeared as strong positive signals. In addition, we observed a loss of MAA signal. No difference in WGA binding was observed between the two cell lines.

Fig. 9. Deconvolution of the ratiometric array data from Fig. 2 into its component red (Cy5) and green (Cy3) channels. Although by eye, the color difference on the ratiometric array for WGA is difficult to distinguish, the numbers clearly show the difference between the Lec8 and Pro-5 samples. The average signal intensities (A.U.) over 10 replicate spots on the array shown above are as follows Cy5 Pro-5 (31,623 ± 877), Cy3 Lec8 (14,678 ± 1,118), Cy5 Lec8 (18,965 ± 1,118), Cy3 Pro-5 (51,502 ± 1,086).

Fig. 10. Single-color glycan analysis of undifferentiated HL-60 cells. Data shown is the average signal over 5 spots with standard deviations. Only positive lectin signals (>1,000) are shown.

Table 1. Print list for lectin microarray

The rough specificities of the lectins are shown (1, 2) (Consortium for Functional Glycomics). Fuc, fucose; Gal, galactose; GalNAc, N-acetyl-D-galactosamine; Glc, glucose; GlcNAc, N-acetyl-D-glucosamine; Man, mannose; Sia, sialic acid.

*Lectins printed on 21-lectin array (CHO samples).

1. Van Damme EJM, Peumans W, Pusztai A, Bardocz S (1998) Handbook of Plant Lectins: Properties and Biomedical Applications (Wiley, New York).

2. Manimala JC, Roach TA, Li Z, Gildersleeve JC (2006) Angew Chem Int Ed 45:3607-3610.

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