Journal: The Journal of Experimental Medicine

Article Title: Accumulation of long-chain fatty acids in the tumor microenvironment drives dysfunction in intrapancreatic CD8 + T cells

doi: 10.1084/jem.20191920

Figure Lengend Snippet: Lipids accumulate in PDA TME while glucose and H6Ps decreases during PDA progression. Related to . (A) Quantification by immunofluorescence of CK19 + area at indicated time points expressed as percentage over the total tissue area. Results are expressed as mean ± SE of three different areas on three independent pancreatic samples. (B) Representative images of Red Oil staining of murine early (left) and late (right) PanINs ( n = 3). Red arrowheads highlight Red Oil–positive areas. Scale bar = 2 mm (left); 0.5 mm (right). (C) Quantification by immunofluorescence of neutral lipids area at indicated time points expressed as percentage of LipidTOX-positive over total tissue or tumor (CK19 + ) area. Results are indicative of three different areas on three independent pancreatic samples. (D) IMS workflow. Frozen pancreata were sectioned at 10-µm thickness on a gold-coated stainless steel target for IMS. IMS was performed on either a 15T or 9.4T SolariX FT-ICR mass spectrometer in positive or negative ion mode from m/z 500 to 2,000 with a 75-µm raster step, generating a mass spectrum at each pixel. The data were segmented in SCiLS through k -means clustering and a segment that best matched CD8 + staining. A mean spectrum was then generated for these segments. The peak list of each segment was searched against the LIPID MAPS database, and mean intensity values for each accurate mass lipid identification were extracted. Through a home-built script in R, data were filtered for visualization by retaining only those lipids with sufficient signal-to-noise ratios and that represented large fold changes between time points. This was done by filtering out log2(intensity) values between −1 and 1 and intensity values <0.3 of the quantile in the control group. Data were then visualized in a heatmap format sorted by head group, fatty acyl chain length, and level of fatty acyl chain unsaturation. (E) Heatmap representations of a series of PEs and PIs showing changes in lipid intensity in KC mice at early and late stages of disease compared with the pancreas of a control mouse. (F–H) Bar plots show the relative fraction of lipids of various FA chain lengths detected at the different disease time points for the indicated lipids (FAs for the three lipid types were summed). The scale for color code is above. (I) Quantification of extracellular glucose in murine early (black) and late (gray) PanINs. Free glucose was measured using a colorimetric-based assay and then normalized based on the weight of the fresh tissue. Each group includes 10 mice. Error bars represent mean ± SEM. Statistics were calculated using an unpaired two-tailed Student’s t test; *, P ≤ 0.05–0.01. (J) IMS intensity distributions on the same tissues presented in show that H6Ps (false-color rainbow images displayed without normalization) decrease moving from earlier to late time points ( n = 2). Scale bar = 2 mm.

Article Snippet: MALDI IMS was performed on either a 15T or 9.4T SolariX FT-ICR mass spectrometer (Bruker Daltonics) equipped with an Apollo dual MALDI/ESI source.

Techniques: Immunofluorescence, Staining, Mass Spectrometry, Generated, Control, Colorimetric Assay, Two Tailed Test

Journal: Rapid communications in mass spectrometry : RCM

Article Title: Protein Identification in Imaging Mass Spectrometry through Spatially Targeted Liquid Micro-Extractions

doi: 10.1002/rcm.8042

Figure Lengend Snippet: A & B) Spectral and imaging data from a 125 μm spatial resolution MALDI protein image from a sagittal section of a mouse pup acquired on a 15T FTICR MS. Four ions were chosen to exemplify the many tissue substructures present as highlighted in the image overlay (B). The proteins sequenced below are highlighted by a star in the average mass spectrum of the protein image. C & D) The high resolution, top-down mass spectrum of m/z 4,898.57 allows for its identification as N-acetylated Thymosin β10. E & F) The high resolution, top-down mass spectrum of m/z 5,675.21 allows for its identification as ATP synthase subunit ε. G & H) The high resolution, top-down mass spectrum of m/z 6,628.37 allows for its identification as N-acetylated histone H2A Type 1. I & J) The high resolution, top-down mass spectrum of m/z 7,513.88 allows for its identification as dimethylated hemoglobin subunit A.

Article Snippet: Ions were generated using a Bruker Captive Spray nanoelectrospray source (Bruker Daltonics, Billerica, MA, USA) and directed into a Bruker SolariX 15T FTICR MS (Bruker Daltonics, Billerica, MA, USA).

Techniques: Imaging