MALDI imaging mass spectrometry is a highly sensitive and selective tool used to visualize biomolecules in tissue. However, identification of detected proteins remains a difficult task. Indirect identifications strategies have been limited by insufficient mass accuracy to confidently link ion images to proteomics data. Here we demonstrate the capabilities of MALDI FTICR MS for imaging intact proteins. MALDI FTICR IMS provides an unprecedented combination of mass resolving power (∼75,000 at m/z 5,000) and accuracy (<5 ppm) for proteins up to ∼12 kDa enabling identification based on correlation with LC-MS/MS proteomics data. Analysis of rat brain tissue was performed as a proof-of-concept highlighting the capabilities of this approach by imaging and identifying a number of proteins including N-terminally acetylated Thymosin β4 (m/z 4,963.502, 0.6 ppm) and ATP Synthase subunit ε (m/z 5,636.074, −2.3 ppm). MALDI FTICR IMS was also used to differentiate a series of oxidation products of S100A8 (m/z 10,164.03, −2.1 ppm), a subunit of the heterodimer calprotectin, in kidney tissue from mice infected with Staphylococcus aureus. S100A8 – M37O/C42O3 (m/z 10228.00, −2.6 ppm) was found to co-localize with bactierial microcolonies at the center of infectious foci. The ability of MALDI FTICR IMS to distinguish S100A8 modifications is critical to understanding calprotectin’s roll in nutritional immunity.

MALDI imaging mass spectrometry is a powerful analytical tool enabling the visualization of biomolecules in tissue. However, there are unique challenges associated with protein imaging experiments including the need for higher spatial resolution capabilities, improved image acquisition rates, and better molecular specificity. Here we demonstrate the capabilities of ultra-high speed MALDI-TOF and high mass resolution MALDI FTICR IMS platforms as they relate to these challenges. High spatial resolution MALDI-TOF protein images of rat brain tissue and cystic fibrosis lung tissue were acquired at image acquisition rates >25 pixels/s. Structures as small as 50 μm were spatially resolved and proteins associated with host immune response were observed in cystic fibrosis lung tissue. Ultra-high speed MALDI-TOF enables unique applications including megapixel molecular imaging as demonstrated for lipid analysis of cystic fibrosis lung tissue. Additionally, imaging experiments using MALDI FTICR IMS were shown to produce data with high mass accuracy (<5 ppm) and resolving power (∼75 000 at m/z 5000) for proteins up to ∼20 kDa. Analysis of clear cell renal cell carcinoma using MALDI FTICR IMS identified specific proteins localized to healthy tissue regions, within the tumor, and also in areas of increased vascularization around the tumor.

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The identification of proteins from tissue specimens is a challenging area of biological research. Many current techniques for identification forfeit some level of spatial information during the sample preparation process. Recently, hydrogel technologies have been developed that perform spatially localized protein extraction and digestion prior to downstream proteomic analysis. Regiospecific protein identifications acquired using this approach have thus far been limited to 1-2 mm diameter areas. The need to target smaller populations of cells with this technology necessitates the production of smaller diameter hydrogels. Herein, we demonstrate hydrogel fabrication processes that allow hydrogel applications down to a diameter of ∼260 μm, approximately 1/15 of the area of previous approaches. Parameters such as the percent polyacrylamide used in hydrogel construction as well as the concentration of trypsin with which the hydrogel is loaded are investigated to maximize the number of protein identifications from subsequent liquid chromatography tandem MS (LC-MS/MS) analysis of hydrogel extracts. An 18% polyacrylamide concentration is shown to provide for a more rigid polymer network than the conventional 7.5% polyacrylamide concentration and supports the fabrication of individual hydrogels using the small punch biopsies. Over 600 protein identifications are still achieved at the smallest hydrogel diameters of 260 μm. The utility of these small hydrogels is demonstrated through the analysis of sub regions of a rat cerebellum tissue section. While over 900 protein identifications are made from each hydrogel, approximately 20% of the proteins identified are unique to each of the two regions, highlighting the importance of targeting tissue subtypes to accurately characterize tissue biology. These newly improved methods to the hydrogel process will allow researchers to target smaller biological features for robust spatially localized proteomic analyses.