Two-color evaluation of Pro-5 vs. Lec8. (a) Examples of N- and O-linked oligosaccharides for Pro-5 and Lec8 cells (31, 32). Carbohydrates are shown in the representational code of the Consortium for Functional Glycomics (www.functionalglycomics.org/static/consortium). (b) Representative microarray data for Pro-5/Lec8 dye-swapped pairs. The two subarrays shown are from the same slide. (c) Plot of the dye-bias-compensated log₂ (Pro-5/Lec8 ratios) for each lectin. Signals in the Cy3 or Cy5 channel were converted to log₂ and then averaged over 10 microarray spots. The mean log₂ (Pro-5 signal) was divided by the mean log₂ (Lec8 signal) for each of the dye pairs [log₂ (Cy5-Pro-5/Cy3-Lec8) and log₂ (Cy3-Pro-5/Cy5-Lec8)] and averaged to remove dye bias (23). Error was propagated by using standard formulas from the standard deviations. Only positive lectin signals are shown (see Materials and Methods). Signals colored in green show ≥2-fold increase in binding to Lec8, whereas signals in red show ≥2-fold better binding to the parent Pro-5. Signals marked in gray display a <2-fold difference in the (Pro-5/Lec8) ratio. (d) Validation of microarray results for Pro-5/Lec8 by means of histology. Cells were fixed in methanol and stained with biotinylated lectins (Con A, GNA, WGA, 80 μg/ml, followed by staining with FITC-labeled avidin). The images were acquired at the same exposure time and are shown on the same scale for every lectin. For comparison, the log₂ (Pro-5:Lec8 ratio) from the histology images for ConA = 0.09 ± 0.27, GNA = −1.64 ± 0.28, and WGA = 3.05 ± 0.41. Ratios were calculated from the images as described in Materials and Methods.

Comparison of all three Lec mutant cell lines. (a) Hierarchical cluster map of ratiometric data for dye-bias-corrected Lec1 hybridized against the common biological reference Pro-5. The heat map was generated and annotated as explained in Fig. 3. Example N- and O-linked glycans for Lec1 are indicated on the left side of the heat map (33). (b) Hierarchical cluster map of ratiometric data for dye-bias-corrected Lec2 hybridized against the common biological reference Pro-5. The heat map was generated as explained previously. Example N- and O-linked glycans for Lec2 are indicated on the left side of the heat map. (c) Hierarchical cluster map of ratiometric data for all three Lec mutants. (d) Comparison between ratiometric data sets and lectin histology for relative HPA binding of Pro-5, Lec1, Lec2, and Lec8 cells. The color bar shows ratiometric data from the microarrays. Cells were stained with biotinylated HPA, followed by FITC-avidin. All fluorescence microscopy images were obtained and displayed by using identical settings.

General schematic of the two-color approach. Cells are lysed and membranes are isolated after sonication as “cellular micellae.” These cellular micellae are then labeled by coupling of either Cy3-NHS or Cy5-NHS with the lysines on proteins. Equivalent amounts (10 μg of protein, ≈10⁵ cells) of the appropriate Cy3- and Cy5-labeled samples are mixed and hybridized to each lectin microarray. After washing, the arrays are scanned by using a GenePix Pro 4100A scanner. The resultant data are extracted by using GenePix Pro 5.1, analyzed, and annotated by using known lectin specificities, deconvolving the array data into carbohydrate patterns.

Heat maps with dendrograms of hierarchical clustering for technical and biological replicates. All heat maps were generated by using Cluster 3.0 (30) and Java TreeView with the Pearson correlation as the distance metric for the arrays with average linkage analysis. The Pearson correlation coefficients are indicated at each branching point. Only the Yang correlations for positive lectins were used in the correlation. Gray boxes represent misprinted lectins that were excluded from the analysis. Misprints were evaluated by examination of the glycoprotein quality control standards. (a) Technical replicates of Pro-5/Lec8. (b) Biological replicates of Pro-5/Lec8.

Differentiation of HL-60 cells into granulocytes causes distinct changes in the glycosylation pattern. Hierarchical cluster map of ratiometric data for differentiated HL-60 cells (Cy3) biological replicates hybridized against a pooled common undifferentiated HL-60 reference (Cy5). Only lectins giving positive signals were used in the cluster analysis (see Materials and Methods). As described previously, the Pearson correlation was used as the distance metric for the arrays with average linkage analysis. Correlation coefficients are shown at select nodes of the dendrogram. In this heat map, green indicates stronger binding to a lectin by the differentiated cells, whereas red indicates stronger binding by the undifferentiated HL-60 cells.