Detection of Antimicrobial Peptides in Stratum Corneum by Mass Spectrometry

Antimicrobial and immunomodulatory peptides (AMPs) are considered as the key players in the maintenance of skin barrier functions. Here, we developed a novel approach for the examination of AMPs in the outermost layer of the epidermis, namely stratum corneum (SC). The SC sample collection by tape stripping was coupled with detection by highly specific and sensitive parallel reaction monitoring (PRM)-based mass spectrometry. We found that hexane-free processing of SC samples produced higher protein yield compared to hexane-based extraction. Of the 18 investigated peptides, 9 could be detected either in healthy or in inflamed skin specimens. Regarding the amount of S100A8, LCN2, LACRT and LYZ significant topographical differences were described among gland poor (GP), sebaceous gland rich (SGR) and apocrine gland rich (AGR) healthy skin regions. We applied a minimally invasive, reproducible approach for sampling, which can be assessed for research and diagnostic purposes and for monitoring the effectiveness of therapies in skin diseases.

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Development of a novel nanoflow liquid chromatography-parallel reaction monitoring mass spectrometry-based method for quantification of angiotensin peptides in HUVEC cultures

PeerJ ◽

10.7717/peerj.9941 ◽

2020 ◽

Vol 8 ◽

pp. e9941

Author(s):

Chuan He ◽

Simiao Hu ◽

Wanxing Zhou

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Culture Supernatant ◽

Reaction Monitoring ◽

Ang Ii ◽

Parallel Reaction ◽

Parallel Reaction Monitoring ◽

Relative Standard ◽

Ang Iv ◽

Nanoflow Liquid Chromatography

Background This study aimed to develop an analytical method using liquid chromatography tandem mass spectrometry (LC-MS/MS) for the determination of angiotensin (Ang) I, Ang (1-9), Ang II, Ang (1-7), Ang (1-5), Ang III, Ang IV in human umbilical vein endothelial cell (HUVEC) culture supernatant. Methods HUVEC culture supernatant was added with gradient concentrations (0.05–1,000 ng/ml) of standard solutions of the Ang peptides. These samples underwent C18 solid-phase extraction and separation using a preconcentration nano-liquid chromatography mass spectrometry system. The target peptides were detected by a Q Exactive quadrupole orbitrap high-resolution mass spectrometer in the parallel reaction monitoring mode. Ang converting enzyme (ACE) in HUVECs was silenced to examine Ang I metabolism. Results The limit of detection was 0.1 pg for Ang II and Ang III, and 0.5 pg for Ang (1-9), Ang (1-7), and Ang (1-5). The linear detection range was 0.1–2,000 pg (0.05–1,000 ng/ml) for Ang II and Ang III, and 0.5–2,000 pg (0.25–1,000 ng/ml) for Ang (1-9) and Ang (1-5). Intra-day and inter-day precisions (relative standard deviation) were <10%. Ang II, Ang III, Ang IV, and Ang (1-5) were positively correlated with ACE expression by HUVECs, while Ang I, Ang (1-7), and Ang (1-9) were negatively correlated. Conclusion The nanoflow liquid chromatography-parallel reaction monitoring mass spectrometry-based methodology established in this study can evaluate the Ang peptides simultaneously in HUVEC culture supernatant.

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