Identification of Hypoxia-Regulated Proteins Using MALDI-Mass Spectrometry Imaging Combined with Quantitative Proteomics

Hypoxia is present in most solid tumors and is clinically correlated with increased metastasis and poor patient survival. While studies have demonstrated the role of hypoxia and hypoxia-regulated proteins in cancer progression, no attempts have been made to identify hypoxia-regulated proteins using quantitative proteomics combined with MALDI-mass spectrometry imaging (MALDI-MSI). Here we present a comprehensive hypoxic proteome study and are the first to investigate changes in situ using tumor samples. In vitro quantitative mass spectrometry analysis of the hypoxic proteome was performed on breast cancer cells using stable isotope labeling with amino acids in cell culture (SILAC). MS analyses were performed on laser-capture microdissected samples isolated from normoxic and hypoxic regions from tumors derived from the same cells used in vitro. MALDI-MSI was used in combination to investigate hypoxia-regulated protein localization within tumor sections. Here we identified more than 100 proteins, both novel and previously reported, that were associated with hypoxia. Several proteins were localized in hypoxic regions, as identified by MALDI-MSI. Visualization and data extrapolation methods for the in vitro SILAC data were also developed, and computational mapping of MALDI-MSI data to IHC results was applied for data validation. The results and limitations of the methodologies described are discussed

MAPPs heat map of identified HLA-DR associated peptides in the HS study.

<p>Heat map visualization of mAb-derived HLA-DR associated peptides for both model antibodies in the HS study. Each sequence position is colored according to the presence and number of different mAb-derived peptides identified. In black colored sequence regions, no peptides were identified, in colored regions, peptides were identified, with the color coding for the number of different peptides identified per position. HS: aggregates generated by heat and shake stress; mAb1: monoclonal antibody 1, mAb2: monoclonal antibody 2, un: unstressed, sl1: stress level 1, sl2: stress level 2.</p

Correlation of HLA-DR associated peptides and peptide clusters measured by MAPPs to the amount of protein present in subvisible particles.

<p>Linear regression analyses of the increase of the HLA-DR associated peptides and clusters as functions of the calculated amount of protein present in the subvisible particles. Left up: HLA-DR associated peptides of mAb1 vs protein amount in subvisible particles (r² = 0.994), left down: HLA-DR associated peptide clusters of mAb1 vs protein amount in subvisible particles (r² = 0.993), right up: HLA-DR associated peptides of mAb2 vs protein amount in subvisible particles (r² = 0.86), right down: HLA-DR associated peptide clusters of mAb2 vs protein amount in subvisible particles (r² = 0.943). HS: aggregates generated by heat and shake stress; FT: aggregates generated by freeze and thaw stress, S: aggregates generated by shear stress mAb1: monoclonal antibody 1, mAb2: monoclonal antibody 2, 1: stress level 1, 2: stress level 2. For further details, please, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086322#s4" target="_blank">Materials and Methods</a>.</p

Results from T-cell activation assay.

<p>T-cell responses determined by IFN-γ ELISpot from donors treated with mAb1, un, sl1 and sl2 material from (A) FT condition (8 donors) and from (B) HS condition (13 donors). The respective heat maps specify to which extent each single donor responded to each treatment. The horizontal line shows the geometric mean of the populations. ***: statistically significant (p<0.001); ns: statistically not significant. HS: aggregates generated by heat and shake stress, FT: aggregates generated by freeze and thaw stress, mAb1: monoclonal antibody 1, SI: Stimulation index, un: unstressed, sl1: stress level 1, sl2: stress level 2.</p

Physicochemical characterization of stressed mAb materials.

<p>Representative MFI screenshots after freeze/thaw (FT) stress of mAb1 (A) unstressed, un; (B) stress level 1, sl1; (C) stress level 2, sl2 and mAb2 un, (E) sl1 and (F) sl2. (G) Particle Size distribution obtained by MFI of mAb1 (left) and mAb2 (right). For visualization the size binning 2–2.5, 2.5–5, 5–10 10–25, 25–50 and 50–400 µm was used. Representative images of individual particles formed by (H) heat/shake, HS; (I) freeze/thaw, FT and (J) shear stress, S. Polydispersity in % (PD%) revealed by DLS for (K) mAb1 and (L) mAb2.</p

Induction of DC maturation by stressed mAb materials.

<p>(A) Percentage of responding donors determined as an at least 1.5-fold upregulation of the response index above unstressed mAb of the maturation markers CD83, CD80 and CD86. (B) Scatter plots of the response indices of individual donors for the measured maturation markers. The dotted line indicates the reference to unstressed mAb at 1.0. The horizontal line represents the average of the individual response indices. HS: aggregates generated by heat and shake stress, FT: aggregates generated by freeze and thaw stress, S: aggregates generated by shear stress, mAb1: monoclonal antibody 1, mAb2: monoclonal antibody 2, RI: Response index, sl1: stress level 1, sl2: stress level 2.</p

Illustration of MAPPs assay procedure and data output example.

<p>A: Illustration of MAPPs assay procedure. Monocytes are isolated from human buffy coats and differentiated to immature DCs in the presence of cytokines. Immature DCs are loaded with the model mAb and induced to maturation with lipopolysaccharide. After 24 hours, mature DCs are frozen. After lysis of mature DCs, HLA-DR:peptide complexes are isolated via immunoprecipitation using anti-HLA-DR coated beads. After several wash steps, peptides are eluted from HLA-DR complexes by pH shift and analysed by nano LC-MS with subsequent sequence identification via SEQUEST database search. B: MAPPs assay data output example. HLA-DR associated peptides can originate from different sequence regions of a protein and can occur in multiple length variants. Peptides in a sample with unique sequence are termed “different peptides”, highlighted with blue box. Nested sets of peptide length variants occurring within a sequence region sharing the same HLA-DR binding core are termed “clusters”, highlighted with green boxes. The number of different peptides per amino acid position can be summarized in a heatmap in which the cell colors are reflecting the number of different peptides, highlighted with red box. Different donors can differ in the pattern of presented peptides depending on binding propensities of their 2 HLA-DR alleles.</p

MAPPs results summary.

<p>Left: Heat map visualization of the number of different peptides and clusters (as sum of heavy and light chain) for both model antibodies mAb1 and mAb2 for all stress conditions S, FT and HS as well as stress levels un, sl1and sl2. Cell colors are reflecting the number of different peptides and clusters. Right: Box and whisker plot of the number of different peptides and clusters for the different mAbs, stress conditions and stress levels. The boxes extend from the 25th to 75th percentiles and the whiskers are plotted from the min to the max value. The median is indicated as a line and the mean as a dot within a box. HS: aggregates generated by heat and shake stress, FT: aggregates generated by freeze and thaw stress, S: aggregates generated by shear stress, mAb1: monoclonal antibody 1, mAb2: monoclonal antibody 2, un: unstressed, sl1: stress level 1, sl2: stress level 2.</p