Methodological Considerations for Lipid and Polar Component Analyses in Human Skin Stratum Corneum

Abstract

Collection of skin very top layer, called stratum corneum, by tape stripping and the analysis of stratum corneum components by mass spectrometry provides multiple advantages for clinical studies that aim to understand the origins of allergic skin diseases and food allergy. However, such a methodology has multiple challenges on the way of complex stratum corneum analysis when molecules of different polarity are needed to be analyzed from minimal amount of skin tape strips. This review provides an overview of current knowledge about lipid and polar molecules in the skin, discusses challenging aspects of sample processing when dealing with skin tape strips, and provides some guidance towards approaches that generate complex, quantitative, normalized to total sample protein data that fit best the purpose of analysis of stratum corneum components for the purpose of clinical trials.

Keywords: Mass spectrometry; Natural moisturizing factor; Skin ceramides; Skin tape strips; Stratum corneum.

Figure 1.. Classification of skin ceramides.

Skin lipids are currently classified into twelve subgroups based on the combination of Long Chain Base (LCB) and the type of N-linked fatty acid (FA). Shown abbreviations are used to designate a specific group of skin ceramides instead of systematic or Lipid Maps designated names to define which group of ceramides is currently discussed. It is estimated that stratum corneum ceramides are comprised of at least 300 different molecular species; however, this number is currently challenged and can be many thousands.

Figure 2.

Skin tape strip processing protocol that allows simultaneous analysis of free lipids, polar components, and protein-bound lipids with data normalization per sample total protein content.

Figure 3.. A survey scan to detect lipids with free hydroxy group (mostly ceramides) in human stratum corneum.

A neutral loss of 18Da scan performed with moderate declustering potential (80V) and low collision energy (20 eV) on lipid extract from human stratum corneum. This survey scan allows identifying major molecular ions for further structural analyses.

Figure 4.. Complexity of human stratum corneum ceramides.

(A) Product ions of the m/z 678.5 demonstrate that C18- and C20-sphingosines and phytosphingosines are the major sphingoid bases in ceramides with the molecular and pseudomolecular ion of the m/z 678.5. This corresponds to structural formulas CER(N26:0)S(18), CER(N24:0)S(20), CER(N26:0)P(18), and CER(N24:0)P(20). Inserts show the nomenclature of MS/MS fragments (left) and the presence of sphingoid bases with odd chain length (C19) (right) within ceramides with the m/z 678.5. (B) Precursor ions scan for the m/z 264.4 demonstrates the presence and the abundance of major NS and AS ceramide as wells as major EOS ceramide species that contain S(18) sphingoid base. Both experiments were performed with declustering potential of 80V and collision energy of 40eV on AB Sciex 6500QTRAP instrument.

Figure 5.. Preferred presence of very long-chain fatty acids in NS ceramides with the longest sphingoid base in human stratum corneum.

Multiple Reaction Monitoring (MRM) profiling of human stratum corneum NS ceramides demonstrates the increasing proportion of 26:0, 28:0, and 30:0-fatty acids within NS ceramides from C18- to C22-sphingosines. Arbitrary horizontal lines delineate approximate “slope” in overall fatty acid hydrophobicity within NS ceramides with different chain length of sphingoid base; vertical line connects NS ceramides with palmitic acid (N16:0). Ceramide detection was achieved in positive ions mode using AB Sciex 6500QTRAP mass spectrometer as a transition from molecular ions to the m/z 264 (S18), m/z 292 (S20), and m/z 320 (S22). HPLC separation was performed using Shimadzu Nexera-X2 UHPLC system on Ascentis Express RP-amide column (2.7 μm 2.1 × 50 mm) with gradient elution from methanol:water:formic acid (50:50:0.5, 5mM ammonium formate) to methanol:chloroform: water:formic acid (90:10:0.5:0.5, 5 mM ammonium formate).

Figure 6.. LC-MS/MS detection of NMF components in human stratum corneum.

Two STS were processed for detection of NMF components and amino acids. LC separation of cis/transUCA, PCA, and other amino acids was achieved using an Acquity UPLC BEH Amide (2.1 × 100 mm, 1.7 μm particle size) column using a gradient from acetonitrile (Solvent A) to methanol:water:formic acid (65:35:0.5, with 5 mM ammonium formate) (Solvent B). All amino acids were detected in positive ions mode using AB Sciex 6500QTRAP instrument. Note the prevalence of the signal for cis-/trans-UCA due to their preferred ionization efficiency at employed chromatographic conditions. Dotted line represents the gradient between solvents A and B (% is shown).