BACKGROUND:Psoriasis is a chronic, immune-mediated inflammatory skin disease. Screening skin metabolites could unravel the pathophysiology of psoriasis and provide new diagnostic approaches. Due to the lack of suitable methodologies for collecting scarce amounts of skin excretions, the psoriatic skin metabolome has not been extensively studied.

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Metabolites excreted by skin have a huge potential as disease biomarkers. However, due to the shortage of convenient sampling/analysis methods, the analysis of sweat has not become very popular in the clinical setting (pilocarpine iontophoresis being a prominent exception). In this report, a facile method for sampling and rapid chemical profiling of skin metabolites excreted with sweat is proposed. Metabolites released by skin (primarily the constituents of sweat) are collected into hydrogel (agarose) micropatches. Subsequently, they are extracted in an online analytical setup incorporating nanospray desorption electrospray ionization and an ion trap mass spectrometer. In a series of reference measurements, using bulk sampling and electrospray ionization mass spectrometry, various low-molecular-weight metabolites are detected in the micropatches exposed to skin. The sampling time is as short as 10 min, while the desorption time is 2 min. Technical precision of micropatch analysis varies within the range of 3-42%, depending on the sample and the method of data treatment; the best technical precision (≤10%) has been achieved while using an isotopically labeled internal standard. The limits of detection range from 7 to 278 pmol. Differences in the quantities of extracted metabolites are observed for the samples obtained from healthy individuals (intersubject variabilities: 30-89%; n = 9), which suggests that this method may have the potential to become a semiquantitative assay in clinical analysis and forensics.

Despite recent advances in phosphopeptide research, detection and characterization of multiply phosphorylated peptides have been a challenge. This work presents a new strategy that not only can effectively extract phosphorylated peptides from complex samples but also can selectively enrich multiphosphorylated peptides for direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Polyarginine-coated diamond nanoparticles are the solid-phase extraction supports used for this purpose. The supports show an exceptionally high affinity for multiphosphorylated peptides due to multiple arginine-phosphate interactions. The efficacy of this method was demonstrated by analyzing a small volume (50 microL) of tryptic digests of proteins such as beta-casein, alpha-casein, and nonfat milk at a concentration as low as 1 x 10 (-9) M. The concentration is markedly lower than that can be achieved by using other currently available technologies. We quantified the enhanced selectivity and detection sensitivity of the method using mixtures composed of mono- and tetraphosphorylated peptide standards. This new affinity-based protocol is expected to find useful applications in characterizing multiple phosphorylation sites on proteins of interest in complex and dilute analytes.