Nanoscale Protein Analysis

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Tau protein is known as a microtubule-stabilizing factor.1 Tau is a microtubule-associated protein (MAP) which is specifically expressed in the neurons, localized in the distal portion of axons. Hyperphosphorylation of tau reduces its affinity to the microtubules and results in aggregation as paired helical filaments (PHFs) and neurofibrillary tangles (NFTs), altering the synaptic connections and leading to neurodegenerative disorders. Aggregation and high phosphorylation of tau are neurotoxic hallmarks of the cell death in Alzheimer’s disease (AD). Though with poorer spatial resolution, it has been shown that it is possible to identify a phosphorylation by measuring the infrared absorption peak of phosphate group observed at about 1070 cm−1 with FTIR method.2 In this application note, IR PiFM chemical mapping will be used to visualize the nanoscale aggregation of tau protein in brain section from transgenic mouse with AD.

Fig 2 - Nanoscale Protein Analysis - App Notes on PiFM
Figure 2: PiFM images at 1744 and 1060 cm−1 highlight the same aggregates of phosphorylated protein.

Figure 1 shows three PiF-IR spectra (light green, blue, and purple) from the sample TG (with AD) and a PiF-IR spectrum from a control sample (without AD); the inset shows the AFM topography of the brain section with the locations from where the spectra were acquired indicated. The light green and blue spectra from TG look similar to the spectrum from the control sample, with prominent amide I (1660 cm−1) and amide II (1550 cm−1) peaks along with less prominent features at 1740, 1243, and 1065 cm−1, which correspond to C=O stretching in the ester functional groups in lipids, PO2 asymmetric stretching in phospholipids, and symmetric stretching in phospholipids, respectively. The purple PiF-IR spectrum acquired on an aggregate-like feature on TG show much stronger peak strengths at 1740, 1243, and 1065 cm−1 with weaker amide peaks. Figure 2 shows PiFM images taken at 1744 and 1060 cm−1 along with the AFM topography of the same region. We can see that the two PiFM images highlight the same features, most likely due to phosphorylation; note that nanoscale features are well reproduced in both images. It is difficult to tell whether the PiFM features are correlated with topographical features. 

Fig 3 - Nanoscale Protein Analysis - App Notes on PiFM
Figure 3: Topography and PiFM images at 1650 and 1740 cm−1 highlighting the surrounding tissue and phosphorylated protein, respectively, on TG (with AD) and control (without AD) samples. Interestingly both the topography and the surrounding tissue look similar despite the preponderance presence of phosphorylated protein aggregates on TG.

Figure 3 shows topography and PiFM images highlighting the surrounding tissue (via amide I, red) and phosphorylated proteins (via the symmetric stretching of PO2, green) of the TG and control samples. AFM topography of the two samples look quite similar with large variations of topographical features, ranging up to a few microns in heights.  Similarly, the features associated with the surrounding tissue (highlighted by the PiFM image at 1650 cm−1) look quite similar. However, the PiFM images taken at 1740 cm−1, highlighting the aggregates of phosphorylated proteins, look strikingly different. For the control sample, the two PiFM images highlight basically the same features, indicating the presence of lipid throughout the tissue. For the sample with AD, the image at 1740 cm−1 highlights not only the lipid/tissue background (most clearly in the upper central portion where topography has less crater-like features) but also large number of aggregates throughout the tissue that we attribute to phosphorylation.

Self-assembled Protein Cage

Self-assembling proteins are candidates as building blocks for “smart” biomaterials that can potentially exploit the rich structural and functional properties of proteins. This has led to the design of new self-assembling protein structures such as these icosahedral protein cages. Since icosahedral protein cages can encapsulate large volumes, they are often used for vaccine design and targeted drug delivery.

In figure 4, three different magnifications (scan sizes) of PiFM images are compared with SEM images of individual self-assembled icosahedral protein cages. The PiFM images were acquired as separate scans in order to check the repeatability of the unusual shape (not quite circular) and signal contrast that occurs within the structure. The sizes and the hexagon-like outline of the shape observed in PiFM images agree well with the SEM images shown below the PiFM images with the same magnification; images are displayed so that the scale bars are equal. PiFM images show contrast within the cage that hints at different facets whose edges and faces exhibit different amide I (1666 cm−1) signal strength; since the excitation light is predominantly polarized along the tip-axis, the image shows polarization dependent response of the amide I band. A schematic drawing of the icosahedral cage is shown next to the PiFM image to highlight the suggested facets in the PiFM image. More details are provided for in reference 3.

Fig 4 - Nanoscale Protein Analysis - App Notes on PiFM
Figure 4: PiFM images of self-assembled protein cages acquired at 1666 cm−1 at three different magnifications along with SEM images. PiF-IR spectrum from a single protein cage clearly shows amide I and amide II peaks.


  1. Cell Biochem Funct. 2020, 38, 686–694
  2. “FT-IR analysis of phosphorylated protein,” Proc. SPIE 5461, Biophotonics New Frontier: From Genome to Proteome, (8 September 2004)
  3. J. Am. Chem. Soc. 2019, 141, 23, 9207-9216

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