Elucidating the molecular landscape of the stratum corneum

Significance Skin is recognized as an intricate assembly of molecular components, which facilitate cell signaling, metabolism, and protein synthesis mechanisms in order to offer protection, regulation, and sensation to the body. Our study takes significant steps to characterize in more detail the complex chemistry of the skin, in particular by generating a better understanding of the uppermost layer, the stratum corneum. Using a state-of-the-art 3D OrbiSIMS technique, we were able to observe the depth distribution, in situ, for a wide range of molecular species. This unprecedented molecular characterization of skin provides information that has the potential to benefit research into fundamental processes, such as those associated with skin aging and disease, and the development and delivery of effective topical formulations.


Supplementary Information Figures
. OrbiSIMS negative polarity spectral data, showing a comparison of the spectra for frozen hydrated (<-170 °C) vs. dehydrated (RT) ex vivo porcine skin tissue. The primary ion beam was Ar3000 + . The total applied ion dose for the frozen hydrated and dehydrated sample was 1.8 x 10 13 and 6.79 x 10 13 respectively. (a) An overview of the spectrum from m/z 100 -700. The ion intensity has been normalized to the total ion count. (b) Three example ion peaks that are absent from the dehydrated spectrum. Specifically, the putatively assigned molecular ions of amino acid threonine (C4H8NO3 -), phospholipid PE 36:2 (C41H77NPO8 -) and ceramide C48:0;O3 (C48H96O4N -). Figure S2. OrbiSIMS negative polarity depth profile data showing the ion intensity variation as function of ion dose/skin depth for the putatively assigned phospholipid species. The primary ion beam was Ar3000 + . The 10 most intense ions are presented. These ions have been used to determine the stratum corneumepidermal boundary, which is approximated with a dotted vertical line. The profile has been compressed using a running average method (100 data points).

Intensity [Arbitrary
Units] Figure S3. OrbiSIMS negative polarity depth profile data showing the ion intensity variation as function of ion dose/skin depth for the putatively assigned triglyceride species. The inset is a zoom in of the region between 2 x 10 12 and 2 x 10 13 ion dose. The primary ion beam was Ar3000 + . The stratum corneumepidermal boundary has been determined, and approximated with a dotted vertical line, based on the ion intensity variation of the phospholipid species ( Figure S2). The profile has been compressed using a running average method (100 data points).

Intensity [Arbitrary
Units] Figure S4. OrbiSIMS negative polarity depth profile data showing the ion intensity variation as function of ion dose/skin depth for the putatively assigned ceramide species. The primary ion beam was Ar3000 + . The stratum corneumepidermal boundary has been determined, and approximated with a dotted vertical line, based on the ion intensity variation of the phospholipid species ( Figure S2). The profile has been compressed using a running average method (100 data points).

Intensity [Arbitrary
Units] Figure S5. OrbiSIMS negative polarity depth profile data showing the ion intensity variation as function of ion dose/skin depth for the putatively assigned fatty acid species. This class of compounds presented two different trends with depth. The primary ion beam was Ar3000 + . The stratum corneumepidermal boundary has been determined, and approximated with a dotted vertical line, based on the ion intensity variation of the phospholipid species ( Figure S2). The profile has been compressed using a running average method (100 data points).

Intensity [Arbitrary
Units] Figure S6. 3D OrbiSIMS negative polarity depth profile data showing the ion intensity variation as function of ion dose/skin depth for the putatively assigned amino acid species. The primary ion beam was Ar3000 + . The stratum corneumepidermal boundary has been determined, and approximated with a dotted vertical line, based on the ion intensity variation of the phospholipid species ( Figure S2). The profile has been compressed using a running average method (100 data points).  Table S6) with sample 1 analyzed in the cryo hydrated state and samples 2 and 3 analyzed in a dehydrated state. The ion intensities have been normalized to the total ion count. The variation of depth distributions of the following compounds was assessed; cholesterol sulfate (C27H45SO4 -), palmitic acid (C16H31O2 -), lignoceric acid (C24H47o2 -), arginine (C6H13N4O2 -), PCA (C5H6NO3 -) and ornithine (C5H11N2O2 -).  Figure S8: OrbiSIMS positive polarity depth profile data showing the ion intensity variation as function of ion dose/skin depth for various putatively assigned compounds. The primary ion beam was Ar3000 + . The ion intensities have been normalized to the total ion count and the profiles have been compressed using a running average method (using a 100 data point average). The stratum corneumepidermal boundary, approximated with a dotted vertical line, has been determined based on the ion intensity variation of the phospholipid species ( Figure S2). Example ions from each identified compound class: PLphospholipids (C5H15NPO4 + ), TGtriglycerides (C7H11 + ), Cerceramides (C39H71O4 + ), FA1 and FA2fatty acids (C18H35O2 + and C26H56NO2 + ) and AAamino acids (C6H15N4O2 + ). The normalized intensity for the phospholipid and amino acid ions have been multiplied by a factor of 10.  Figure S9. Data produced from 3D OrbiSIMS negative polarity depth profile analysis of frozen hydrated ex vivo skin, both human and porcine. The primary ion beam was Ar3000 + . Depth profiles for both (i) human and (ii) porcine samples, showing the ion intensity variation as function of ion dose/skin depth for various putatively assigned compounds. The ion intensities have been normalized to the total ion count and the profiles have been compressed using a running average method (using a 100 data point average). The stratum corneumepidermal boundary, approximated with a dotted vertical line, has been determined for each species based on the ion intensity variation of the phospholipid ion. Example ions from the following compound classes are shown: PLphospholipids (C41H77NPO8 -), Cerceramides (C44H86NO5 -), FA1 and FA2fatty acids (C27H47O2and C16H31O2 -), AAamino acids (C5H11N2O2 -). The normalized intensity for the porcine ceramide and phospholipid ions has been multiplied by a factor of 100 and 1000 respectively.

Epidermis SC
Figure S10. 3D OrbiSIMS negative polarity depth profile data showing the ion intensity variation as function of ion dose/skin depth for the putatively assigned exogenous compound sodium lauryl sulfate (C12H25SO4 -). The primary ion beam was Ar3000 + . The stratum corneumepidermal boundary has been determined, and approximated with a dotted vertical line, based on the ion intensity variation of the phospholipid species ( Figure S2). The ion intensity has been normalized to the total ion count and the profile has been compressed using a running average method (100 data points).  * Without further investigation we were unable to distinguish whether the structure related to the ceramide or the acyl-ceramide.