Determining the N-Glycan and Collagen/Extracellular Matrix Protein Compositions in a Novel Outcome Cohort of Prostate Cancer Tissue Microarrays Using MALDI-MSI

Abstract

Using matrix-assisted laser desorption/ionization mass spectrometry imaging techniques on a unique cohort of prostate cancer tissues, we highlighted several molecular characteristics of matrix that have potential to act as early predictors of prostate cancer metastasis.

Figure 1

Workflow protocols for sample preparation. For N-glycan analysis, FFPE slides were deparaffinized and rehydrated before undergoing antigen retrieval in citraconic buffer. PNGaseF (1) was applied as a “dry” molecular coating before a 2-hour incubation at 37°C. CHCA matrix was applied, and imaging of N-glycans was performed. For analysis of core- vs outer-arm fucosylated N-glycans, enzyme EndoF3 (2) was dialyzed before application, and imaging of EndoF3-digested N-glycans was performed before subsequent PNGaseF digestion and imaging of remaining N-glycans. Following glycan imaging, CHCA matrix was removed from tissues, and samples were histologically analyzed following H&E staining. Tissues underwent a second antigen retrieval with tris buffer before COLaseIII (3) was applied as a “dry” molecular coating. Slides were incubated for 5 hours at 37°C. CHCA matrix was applied, and imaging of collagen/ECM peptides was performed.

Figure 2

Glycan imaging data from TMA comparing metastasis vs. no recurrence. A, MALDI-MSI images of TMAs from metastasis (MET) category and no recurrence (NED) category. N-glycan images representative of differentiating expression of select N-glycan categories, followed by segmentation analysis of 152 detected N-glycans. B–I, Box plot and ROC curves for each category of N-glycan detected within each group of TMAs. t test results shown on each boxplot.

Figure 3

Statistical analysis of detected N-glycans in each category of TMA. A, PCA score plot of all 152 N-glycans. B, PLS-DA plot and corresponding importance feature plot. C,t test plot of NED vs. MET with structures shown for the top 10 most significant peaks. D, Pattern search graph showing the top 25 peaks correlated with 2905.0368 (Hex8dHex1HexNAc7), m/z values of N-glycans with at least one or more fucose shown in red boxes. VIP, variable importance in projection.

Figure 4

Statistical analysis of detected N-glycans in each category of TMA following EndoF3 and PNGaseF digestion. A,t test plot of detected N-glycans following EndoF3 digestion. Structures shown for top 10 most significant N-glycans (B) t test plot of detected N-glycans following PNGaseF digestion. Structures shown for top 10 most significant N-glycans C, Left-Right: PCA, PLS-DA plots for detected N-glycans following EndoF3 digestion. ROC curve showing NED vs. MET after EndoF3 digestion D, Left-Right: PCA, PLS-DA plots for detected N-glycans following PNGaseF digestion. ROC curve showing NED vs. Mets after PNGaseF digestion.

Figure 5

Collagen/ECM imaging data from TMAs comparing metastasis vs. no recurrence. A, Representative MALDI-MSI images of TMAs from metastasis (MET) category and no recurrence (NED) category. B, Segmentation analysis of 294 detected peptides. C, Volcano plot created from list of 294 detected peptides D, Heatmap of top 150 significantly different detected peptides. E, Box plot and ROC curve for 1179.5603 m/z. F, Box plot and ROC curve for 1426.6743 m/z. G, Box plot and ROC curve for 890.4250 m/z.

Figure 6

Analysis of collagen/ECM profile of NED vs. MET cohort from data obtained by LC-MS/MS. A, Pie chart representations of abundance (peak intensity and number of peptides identified via LC-MS/MS) of all detected protein species within the MET cohort. B, Pie chart representations of abundance of all detected protein species within the NED cohort.

Figure 7

Collagen/ECM MALDI-MSI data from full tissue samples (A) H&E-stained images of tissue from metastasis (MET) category and no recurrence (NED) category. Region where TMA was sampled is circled in black. Note that the MET tissue is the same H&E image used in Supplementary Fig. S8A, as N-glycans and collagenase peptides were detected sequentially on the same tissue slide. B, Corresponding MALDI-MSI images after digestion with COLaseIII from the two metastasis (MET) tissues (top) and the two no recurrence (NED) tissues (bottom). Corresponding m/z values for each image are shown to the right. C, Representative MALDI-MSI images from the metastasis (MET) TMA and no recurrence (NED) TMA of the same mass corresponding to full tissues. Note that the collagenase peptide image for m/z = 891.4323 is the same data image used in Fig. 5A for this ion.