Use of lipidomics to investigate sebum dysfunction in juvenile acne

Use of lipidomics to investigate sebum dysfunction in juvenile acne

June 2016

Camera E, Ludovici M, Tortorella S, Sinagra JL, Capitanio B, Goracci L, Picardo M.


Acne is a multifactorial skin disorder frequently observed during adolescence with different grades of severity. Multiple factors centering on sebum secretion are implicated in acne pathogenesis. Despite the recognized role of sebum, its compositional complexity and limited analytical approaches have hampered investigation of alterations specifically associated with acne. To examine the profiles of lipids distribution in acne sebum, 61 adolescents (29 males and 32 females) were enrolled in this study. Seventeen subjects presented no apparent clinical signs of acne. The 44 affected individuals were clinically classified as mild (13), moderate (19), and severe (12) acne. Sebum was sampled from the forehead with SebutapeTM adhesive patches. Profiles of neutral lipids were acquired with RR-RP/HPLC-TOF/MS in positive ion mode.  

Univariate and multivariate statistical analyses led to the identification of lipid species with significantly different levels between healthy and acne sebum. The majority of differentiating lipid species were diacylglycerols, followed by fatty acyls, sterols, and prenols. Overall, the data indicated an association between the clinical grading of acne and sebaceous lipid fingerprints and highlighted diacylglycerols as more abundant in sebum from adolescents affected with acne. 

Peptide metabolism: Identification

Peptide metabolism: Identification of Metabolite structures of GLP-1 receptor agonists in different in-vitro systems using high resolution mass spectrometry

64th ASMS Conference on Mass Spectrometry and Allied Topics, San Antonio, TX (United States of America) … 05 June 2016 




Peptide drugs are an important class of therapeutics under investigation in various pharmaceutical companies. Assessment of peptide stability in vitro, the identification of cleavage sites and structure elucidation of degradation products are important tasks of drug metabolism scientists. However, most in vitro systems established to investigate metabolism of small molecules (e.g., microsomes) are not relevant for peptides because most peptides show low cell membrane permeability and are subject to hydrolysis by enzymes expressed on epithelial cell surfaces. In addition to relevant in vitro systems, appropriate mass spectrometry approaches and tailored software tools are required due to the higher molecular weight, presence of multiple-charge stages upon electrospray ionization and increasing molecular complexity (modified amino acids, cyclisation etc.) of peptide drug candidates.  



Glucagon-Like Peptide-1, (GLP-1) and three analogs (taspoglutide, liraglutide, exenatide) were incubated with the human recombinantly expressed enzymes dipeptidyl-peptidase-IV (DPP-4) and neutral endopeptidase (NEP) as well as with various cellular systems, namely primary and immortalized human umbilical vein endothelial cells (HUVEC cells), TERT1-immortalized renal proximal tubule epithelial cells (RPTECs-TERT1 cells) and human hepatocytes. Samples were analyzed up to 24 hours using a Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer in data dependent scan mode. The mass inclusion list set-up (up to z = 5) and the post-acquisition data analyses were performed using the recently introduced peptide mode integrated in MassMetaSite (MMS 3.2.0). MMS extracted metabolite peaks and interpreted MS/MS fragmentation to provide structural proposals. The results were reviewed using WebMetabase (version 3.1.4).  


Preliminary Data  

The peptide mode of the MassMetaSite software was successfully applied to process full scan HRMS data to detect and identify metabolites of 4 model peptides. MMS proposed definitive metabolite structures to the identified metabolite peaks based on the interpretation of MS2 fragmentation data. Based on these metabolite structures, peptide bond cleavage sites could be demonstrated. WebMetabase was able to sort and match metabolites based on retention time and MS2 fragmentation across the different in vitro experiments resulting in an efficient workflow to compile results for comparison of different in vitro systems regarding metabolites formed. The results showed that GLP-1 was metabolized rapidly in the presence of DPP-4 (t1/2  7 min). The main metabolite identified by MMS (< 2 ppm) resulted from N-terminal cleavage after amino acid 8 (Ala), corresponding to GLP-1 (9-37). This observation was in line with reports from literature.  

The same metabolite increased with time in incubates with primary HUVEC, immortalized HUVEC and hepatocytes indicative of functional DPP-4 activity in these cell lines. Turnover in presence of DPP-4 was as well seen for taspoglutide and liraglutide, however at a slower rate compared to GLP-1. Analog to GLP-1 the cleavage sites were assigned after amino acid 8 at the N-terminus resulting in liraglutide (9-37) and taspoglutide(9-37). Taspoglutide in presence of NEP was initially cleaved between the amino acids Ser18-Tyr19 and Tyr19-Leu20 position (analog for liraglutide).

Further peptidic cleavage lead to shorter peptides mainly seen in human hepatocytes. For taspoglutide and liraglutide, mostly the same metabolites were seen in HUVEC cells when compared with the isolated enzyme systems NEP and DPP-4 alone. Preliminary data suggest that no significant qualitative difference was observed between primary and immortalized HUVEC cells for degradation products of GLP-1 and its structural analogs. In conclusion this approach might be used for peptide metabolism investigations. Novel Aspect New software-aided approach to analyze HRMS data to investigate stability and cleavage sites of peptides in different in vitro systems. 

Enhancing throughput of glutathione adduct formation studies and structural identification using a software-assisted workflow based on high resolution mass spectrometry (HRMS) data 

Enhancing throughput of glutathione adduct formation studies and structural identification using a software-assisted workflow based on high resolution mass spectrometry (HRMS) data 

October 2016.

E.N. Cece-Esencan, F. Fontaine, G. Plasencia, M. Teppner, A. Brink, A. PahlerI. Zamora.


The bioactivation of drugs to Reactive Metabolites (RM) has been related to drug-induced liver injury and hypersensitivity reactions in patients. Therefore, many pharmaceutical companies are investigating the potential to form reactive metabolites in vitro as an integral part of the optimization of drug candidates. A computerassisted workflow to efficiently analyze larger numbers of compounds for the formation of glutathione trappable RM is presented here. A set of 95 compounds with known bioactivation potential was selected for this study.

Incubations with human liver microsomes were prepared with GSH. The acquisition of MS/MS spectra was triggered by ion intensity. MS with singly and doubly charged ions were used for peak detection and MS/MS spectra were used for structural elucidation. A confidence classification system for the GSH peak detection (high, medium, low) was developed based on the detection of characteristic fragment ions or neutral losses and applied to remove potential false positive results. A comparative analysis of the HRMS results with literature data was carried out.

The most frequently observed Neutral Loss (NL) found in singly charged GSH adducts (drug-glutathione conjugates) were, the Neutral Loss (NL, 129 Da) and Fragment Ion (FI, m/z 308) and in the doubly charged ones the Fragment Ion (FI, m/z 130). These NL and FI were used to identify GSH-related drug metabolites. MS/MS spectra were inspected to aid structural elucidations: 17% of drug substrates and 29 % of GSH adduct metabolites were identified with only doubly charged ions, stressing the importance of considering this charge state in the identification workflow.

A total of 41 compounds that form GSH adducts were retrieved from literature (HRMS, identified 28 compounds (68%) in high confidence, and the same result was obtained using precursor ion scan). By the confidence analysis of GSH peaks, the quality of the each GSH adduct was determined. 

Software-aided cytochrome P450 reaction phenotyping and kinetic analysis in early drug discovery

Software-aided cytochrome P450 reaction phenotyping and kinetic analysis in early drug discovery

January 2016.

Cece-Esencan EN; Fontaine F; Plasencia G; Teppner M; Brink A; Pähler A; Zamora I


Rationale: Cytochrome P450 (CYP450) reaction phenotyping (CRP) and kinetic studies are essential in early drug discovery to determine which metabolic enzymes react with new drug entities. A new semi-automated computer-assisted workflow for CRP is introduced in this work. This workflow provides not only information regarding parent disappearance, but also metabolite identification and relative metabolite formation rates for kinetic analysis.

Methods: Time-course experiments based on incubating six probe substrates (dextromethorphan, imipramine, buspirone, midazolam, ethoxyresorufin and diclofenac) with recombinant human enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) and human liver microsomes (HLM) were performed. Liquid chromatography/high-resolution mass spectrometry (LC/HRMS) analysis was conducted with an internal standard to obtain high-resolution full-scan and MS/MS data. Data were analyzed using Mass-MetaSite software. A server application (WebMetabase) was used for data visualization and review.

Results: CRP experiments were performed, and the data were analyzed using a software-aided approach. This automated-evaluation approach led to (1) the detection of the CYP450 enzymes responsible for both substrate depletion and metabolite formation, (2) the identification of specific biotransformations, (3) the elucidation of metabolite structures based on MS/MS fragment analysis, and (4) the determination of the initial relative formation rates of major metabolites by CYP450 enzymes.

Conclusions: This largely automated workflow enabled the efficient analysis of HRMS data, allowing rapid evaluation of the involvement of the main CYP450 enzymes in the metabolism of new molecules during drug discovery.

Development of higher-thoughput metabolic soft spot assay with integrated assessment of Glutathione adduct formation

Development of higher-thoughput metabolic soft spot assay with integrated assessment of Glutathione adduct formation

64th ASMS Conference on Mass Spectrometry and Allied Topics, San Antonio, TX (United States of America) 05 June 2016 


Introduction High clearance due to extensive metabolism by cytochrome P450 is a common ADME liability of discovery-stage compounds. To provide structural information to facilitate ADME optimization, we have established a higher-throughput in vitro soft spot identification assay using HRMS/MS, sample pooling and software-assisted structure elucidation, as reported previously1. In this work, we present an additional improvement to the assay by integrating the assessment of glutathione (GSH) adduct formation, which could indicate bioactivation to reactive metabolites, in an enhanced analysis and data-review workflow. Methods A dual-concentration (30 and 0.5 µM) incubation approach was used, with liver microsomes that were supplemented by both NADPH and GSH. A Shimadzu Nexera HPLC system and a Thermo Q Exactive tandem mass spectrometer were used for LC-MS analysis. A 10-min gradient was used with an Agilent Eclipse+ C18 column and a mobile phase of 0.2% formic acid in water and acetonitrile. During analysis of the 30 µM samples, the mass spectrometer was operated with alternating full scans and data dependent scans with an inclusion list generated automatically by Mass-MetaSite. For the 0.5 µM samples, only full scan data was collected. Software-assisted soft spot ID was performed using Mass-MetaSite, and semi-quantitation of metabolites was performed using GMSU/QC software. Preliminary Data The use of an inclusion list of both singly and doubly charged masses generated by Mass-MetaSite for the data dependent acquisition ensured the collection of important MS/MS spectra for potential metabolites and GSH adducts. In addition, all-ion fragmentation data was acquired following a separate injection in case an unexpected metabolite failed to trigger MS/MS spectrum acquisition. All data from the 30 µM samples was processed by Mass-MetaSite software, which compared chromatograms to identify metabolites and then assigned structures by comparing their theoretical and experimental MS/MS spectra in an unattended fashion. After major metabolites were assigned by Mass-MetaSite, their accurate masses were imported into GMSU/QC software for automatic peak extraction and integration, and %-remaining vs. time plots of metabolites detected in the 0.5 µM samples were generated automatically. Using this workflow, we established the feasibility of a higher-throughput assay for soft-spot identification with integrated assessment of GSH adduct formation, at a per batch capacity of 8 individual compounds in liver microsomes from 2 species. The results obtained from this workflow were consistent with those previously reported. The detailed assay incubation, data acquisition and data processing workflow, as well as results from literature compounds will be presented. 1 Paiva A, Klakouski C, Zvyaga T, Johnson B, Josephs J, Humphreys WG, Weller H and Shou WZ, “Optimization of a High-throughput Metabolic Soft Spot Assay with Pooled Sample Analysis and Software-assisted Structure Elucidation,” presented at the 61st Annual Conference of American Society for Mass Spectrometry (ASMS) on Mass Spectrometry and Allied Topics, Minneapolis, Minnesota, 2013. Novel Aspect The integrated assessment of glutathione adduct formation in a high-throughput metabolic soft spot assay.