Tissue images and MS data were provided courtesy of Jessica Moore, and Jeff Spraggins, Mass Spectrometry Research Center, Department of Chemistry, Vanderbilt University School of Medicine, Nashville, TN, USA.
Application & Background
Figure 1. Adapted from (3), inhibition of bacterial processes through Mn2+ chelation by calprotectin. At the site of infection, neutrophils deliver a “double hit” to S. aureus by releasing calprotectin (crosses), which chelates Mn2+ and Zn2+, thus sensitizing S. aureus to ROS generated by the neutrophil.
MALDI mass spectrometry imaging (MSI), allows for the visualization of biomolecules in tissues in addition to specific molecular identification and localization that can greatly expand the typical information of gained from traditional histological and western blotting techniques. Specifically, MALDI MSI of intact proteins allows for many of protein isoforms (proteoforms) to be identified and spatially resolved (1). Confirmation of protein identity remains to be one of the biggest challenges in proteomic research, with several strategies developed including bottom-up, top-down and indirect identification.
Immune cells have the capacity to attack pathogens via a wide range of strategies, including reactive oxygen species (ROS) generation and the chelation of metals that are essential for bacterial proliferation and antioxidant defense (2). Calprotectin is a Zn- and Mn-binding protein heterodimer, highly abundant in neutrophils, with antimicrobial and antifungal properties based on the sensitization of the bacterium to superoxide oxidative damage (3). In the infectious foci, calprotectin is in close proximity to ROS-generating proteins, thereby being exposed to high levels of oxidative stress. Understanding the consequences of oxidative modifications on protein function of S100A8 is of high relevance in order to gain insights into host-pathogen interactions. In this study, we aimed to image the various proteoforms of a particular subunit of calprotectin (S100A8), in kidney tissue from mice infected with Staphylococcus aureus.
Experimental
Sample Preparation
Table 1. Spray parameters MALDI matrix deposition.
10 μm cryosections of mouse kidney were placed onto conductive ITO coated slides (Delta Technologies). Tissue was then washed with 70% EtOH for 30 sec, 100% EtOH for 30 sec, Carnoy fluid (6:3:1 EtOH: chloroform: acetic acid) for 2 min, 100% EtOH for 30 sec, H2O with 0.2% TFA for 30 sec, and 100% EtOH for 30 sec, and stored at -80 °C until IMS analysis. Tissue sections were sprayed using an HTX TM-Sprayer using parameters detailed in Table 1.
MALDI Mass spectrometry imaging
Images were collected with a laser setting of ~50 μm and a pixel spacing of 75 μm in x and y axis, over a mass range of m/z 1,000 to 15,000. Calibration was performed externally using CsI clusters.
Results
Figure 2. MALDI FTICR IMS of intact proteins from mouse kidney tissue of wild-type (blue), or calprotectin-KO (pink), infected with S. aureus. Images were taken from a lesion where neutrophils accumulate, in positive ion mode.
Figure 2 shows MALDI FTICR IMS of intact proteins obtained from a mouse kidney infected with S. aureus. Data were collected with a resolving power of ~75,000 at m/z 5,000, resulting in 2552 peaks detected between m/z 2,000 and 12,000. Ions up to m/z ~12,000 were detected with high sensitivity. The average mass spectrum of the entire data set for a wild type (Wt), and a calprotectin-knock-out (KO) mouse infected with S. aureus are shown in Figure 2A. The protein subunit S100A8 from the heterodimer calprotectin was detected at m/z 10,1643.03 ([M+H]+1, -2.1 ppm) only in the Wt, and was identified by top-down fragmentation. A closer view within the m/z range 10,160 – 10,320 allowed to identify several potential proteoforms of S100A8. Fig. 2B shows the S100A8 peak, as well as two of its oxidation products S100A8+O (m/z 10,180.07, [M+H]1+), and S100A8+4O (m/z 10,228.00, [M+H]1+).
Figure 3. Selected MALDI FTICR MS ion images of intact proteins from mouse kidney infected with S. aureus. m/z 8,564 (ubiquitin, yellow), m/z 4,963 (thymosin 4, green), m/z 5,653 (histone H4, grey), m/z 7,024 (histone H2A1, blue), m/z 10,164 (S100A8, red). ~75 µm spatial resolution. Acquisition time ~4 hrs. Scale bar 2 mm.
A traditional H&E stain of the tissue is presented in Figure 3A for spatial correlation. The ion images for a series of biologically-relevant proteins (ubiquitin, thymosin, histones H4 and H2A1) and S100A8 are shown in Figure 3B. Please note the higher localization of S100A8 in the infectious loci.
Representative ion images for S100A8 and one post-translational oxidation product are shown in Figure 4A and B, respectively. By comparing the H&E stained micrograph with the MALDI FTICR IMS overlay data (Figure 4D), the oxidative form of S100A8 shows to accumulate near the center of the infectious foci, suggesting highly oxidative processes occur at the host-pathogen interface.
Figure 4. Selected MALDI FTICR MS ion images of (A) intact S100A8 (red, m/z 10,164) and (B) its oxidation product S100A8+4O (blue, m/z 10,228) from mouse kidney infected with S. aureus. (C) Shows the H&E stain, same as Fig 3A. (D) Two color overlay; m/z 10,164 (red), m/z 10,228 (blue). ~75 µm spatial resolution. Acquisition time 2 sec / pixel, total ~4 hrs. Scale bar 2 mm.
Figure 5. LC-MS/MS results for S100A8 – M37O/C42O3. High-mass resolution bottom-up fragmentation data for the tryptic peptide shown above.
Finally, to confirm that the observed ion at m/z 10,228.00 is an oxidation proteoform of S100A8, as well as to identify the specific AA residues oxidized, top-down and bottom-up strategies were employed for identification (Figure 5). The tryptic peptide shown in Fig 5 was bottom-up fragmented and presented oxidation of the methionine 37 and cysteine 42 residues.
MALDI FTICR IMS experiments such as the ones presented in this study are highly valuable for immunological biology given that post-translational modification of proteins (i.e. oxidation) play a key role in the immune response.
Conclusions
Protein identification coupled to MALDI IMS has remained a significant challenge, due to the limited mass accuracy and low resolving power. MALDI FTICR IMS significantly advances he field of molecular imaging by combining MALDI IMS and LC-MS/MS at their optimum performance.
References
(1) Smith, L. M., Kelleher, N. L., T h e Consortium for Top Down Proteomics, Proteoform: a single term describing protein complexity. Nat. Methods 10, 186–187 (2013)
(2) Russell, D.G.: Staphylococcus and the healing power of pus. Cell Host Microbe 3, 115 –116 (2008)
(3) Hood, M. I., Skaar, E. P.: Nutritional immunity: transition metals at the pathogen-host interface. Nat. Rev. Microbiol. 10, 525 –537 (2012)