MALDI imaging mass spectrometry profiling of proteins and lipids in clear cell renal cell carcinoma

Abstract

Reducing the incidence and mortality rates for clear cell renal cell carcinoma (ccRCC) remains a significant clinical challenge with poor 5-year survival rates. A unique tissue cohort was assembled of matched ccRCC and distal nontumor tissues (n = 20) associated with moderate risk of disease progression, half of these from individuals who progressed to metastatic disease and the other half who remained disease free. These tissues were used for MALDI imaging MS profiling of proteins in the 2-20 kDa range, resulting in a panel of 108 proteins that had potential disease-specific expression patterns. Protein lysates from the same tissues were analyzed by MS/MS, resulting in identification of 56 proteins of less than 20 kDa molecular weight. The same tissues were also used for global lipid profiling analysis by MALDI-FT-ICR MS. From the cumulative protein and lipid expression profile data, a refined panel of 26 proteins and 39 lipid species was identified that could either distinguish tumor from nontumor tissues, or tissues from recurrent disease progressors from nonrecurrent disease individuals. This approach has the potential to not only improve prognostic assessment and enhance postoperative surveillance, but also to inform on the underlying biology of ccRCC progression.

Keywords: Biomedicine; Lipid profiling; MALDI imaging; MALDI-FT-ICR; Renal cancer; Tissue proteomics.

Figure 1. Representative MALDI-MS images of proteins in matched ccRCC tumor and non-tumor tissues

A. Three images of ions (m/z = 4953, 10164, 13904) with increased expression in non-recurrent tumor tissues (114NR-T). H&E stained images of both tissues are also shown, including representative magnifications of regions at 4×, 10× and 20× magnification, indicated by the inset boxes. B. Two images of two ions at m/z = 4901 and m/z = 4967 enriched in tumor for a non-recurrent pair (20NR), and a m/z = 4967 with higher tumor expression. C. Representative images of increased detection of m/z= 15043 and 13930 in a non-tumor/tumor pair.

Figure 1. Representative MALDI-MS images of proteins in matched ccRCC tumor and non-tumor tissues

A. Three images of ions (m/z = 4953, 10164, 13904) with increased expression in non-recurrent tumor tissues (114NR-T). H&E stained images of both tissues are also shown, including representative magnifications of regions at 4×, 10× and 20× magnification, indicated by the inset boxes. B. Two images of two ions at m/z = 4901 and m/z = 4967 enriched in tumor for a non-recurrent pair (20NR), and a m/z = 4967 with higher tumor expression. C. Representative images of increased detection of m/z= 15043 and 13930 in a non-tumor/tumor pair.

Figure 2. Heat map of 108 selected protein peaks

Protein peak intensities for a representative region of interest for each sample (n=20) were normalized and average peak intensities were log10 transformed before plotting as a heatmap. The scale is log10 average normalized peak intensity. Also, a Wilcoxon signed-rank or rank-sum test was used to compare peak intensities between tumor versus normal and recurrent versus non-recurrent, respectively (Supplemental Table 2).

Figure 3. Representative MALDI-MS images of lipid species in matched ccRCC tumor and non-tumor tissues

Image profiles of ten lipids are shown for two matched tissues, a non-recurrent (20NR) and recurrent (238R) tumor/non-tumor pair. The representative lipids were selected as part of the 39 lipids that were most discriminatory across conditions (Supplemental Table 4). A. Five lipid ions that are predominantly over-expressed in non-tumor tissues. B. Five lipid ions that are predominantly expressed in tumor tissues.

Figure 4. Principal Component Analysis of Protein and Lipid Species

Protein and lipid peaks of interest were selected based on an individual peak's ability to distinguish groups (AuROC > 0.70) and statistical significance (p < 0.05; Supplemental Tables 2 and 4), then evaluated separately and combined using PCA. A. Plot of PCA results using 26 proteins. B. Plot of PCA results using 39 lipids. C. Plot of PCA results using combined 26 proteins and 39 lipids.