Protocol for the Isolation of Stratum Corneum from Pig Ear Skin: Evaluation of the Trypsin Digestion Conditions

Stratum corneum (SC) represents the outermost layer of the skin, being an effective barrier against the entry of molecules and pathogens. Skin research has given particular focus to SC as it hampers effective drug delivery for cosmetical and therapeutical purposes. Following recommendations to develop alternative models to animals, the SC isolated from skin obtained from medical procedures or from pigs has gained extensive attention. Yet, there is still missing a standard and simple procedure accepted within the scientific community to avoid application of different isolated SC methodologies, a fact that may hamper progress in skin research. Considering this challenge, the present study evaluated different experimental conditions aiming to establish a useful and sustainable solvent-free procedure for the obtention of a realistic SC model. The studied trypsin digestion parameters included concentration, incubation period and temperature. Isolated SC was characterized using histological analysis and calcein’s permeability, after the procedure and during a 6-week storage period. Data recommend trypsin digestion at 4 °C for 20 h as the most effective procedure to isolate SC from pig ear skin. This work contributes to standardize the SC isolation procedure, and to obtain a valuable and reliable SC mimetic model for skin drug development.

Mimicking LysC Proteolysis by ‘Arginine-Modification-cum-Trypsin digestion’: Comparison of Bottom-Up & Middle-Down Proteomic Approaches by ESI-QTOF-MS

Background: Middle-down (MD) proteomics is an emerging approach for reliable identification of post- translational modifications and isoforms, as this approach focuses on proteolytic peptides containing > 25 - 30 amino acid residues (a.a.r.), which are longer than typical tryptic peptides. Such longer peptides can be obtained by AspN, GluC, LysC proteases. Additionally, some special proteases were developed specifically to effect MD approach, e.g., OmpT, Sap9, etc. However, these proteases are expensive. Herein we report a cost-effective strategy, ‘arginine modification-cum trypsin digestion’, which can produce longer tryptic peptides resembling LysC peptides derived from proteins. Objective:: To obtain proteolytic peptides that resemble LysC peptides, by using 'trypsin', which is an less expensive protease. Methods: This strategy is based on the simple principle that trypsin cannot act at the C-termini of those arginines in proteins, whose sidechain guanidine groups are modified by 1,2-cyclohexanedione or phenylglyoxal. Results: As a proof of concept, we demonstrate this strategy on four models: -casein (bovine), - lactoglobulin (bovine), ovalbumin (chick) and transferrin (human), by electrospray ionization-mass spectrometry (ESI-MS) involving hybrid quadrupole time-of-flight. From the ESI-MS of these models, we obtained several arginine modified tryptic peptides, whose lengths are in the range, 30 - 60 a.a.r. The collision-induced dissociation MS/MS characteristics of some of the arginine modified longer tryptic peptides are compared with the unmodified standard tryptic peptides. Conclusion: The strategy followed in this proof-of-concept study, not only helps in obtaining longer tryptic peptides that mimic LysC proteolytic peptides, but also facilitates in enhancing the probability of missed cleavages by the trypsin. Hence, this method aids in evading the possibility of obtaining very short peptides that are < 5 - 10 a.a.r. Therefore, this is indeed an cost-effective alternative/substitute for LysC proteolysis and in turn, for those MD proteomic studies that utilize LysC. Additionally, this methodology can be fruitful for mass spectrometry based de novo protein and peptide sequencing.

Structural analysis of the GPI glycan

Glycosylphosphatidylinositol (GPI) anchoring of proteins is an essential post-translational modification in all eukaryotes that occurs at the endoplasmic reticulum (ER) and serves to deliver GPI-anchored proteins (GPI-APs) to the cell surface where they play a wide variety of vital physiological roles. This paper describes a specialized method for purification and structural analysis of the GPI glycan of individual GPI-APs in yeast. The protocol involves the expression of a specific GPI-AP tagged with GFP, enzymatic release from the cellular membrane fraction, immunopurification, separation by electrophoresis and analysis of the peptides bearing GPI glycans by mass spectrometry after trypsin digestion. We used specifically this protocol to address the structural remodeling that undergoes the GPI glycan of a specific GPI-AP during its transport to the cell surface. This method can be also applied to investigate the GPI-AP biosynthetic pathway and to directly confirm predicted GPI-anchoring of individual proteins.

Rapid Differential Detection of Abrin Isoforms by an Acetonitrile- and Ultrasound-Assisted On-Bead Trypsin Digestion Coupled with LC-MS/MS Analysis

The high toxic abrin from the plant Abrus precatorius is a type II ribosome-inactivating protein toxin with a human lethal dose of 0.1–1.0 µg/kg body weight. Due to its high toxicity and the potential misuse as a biothreat agent, it is of great importance to developing fast and reliable methods for the identification and quantification of abrin in complex matrices. Here, we report rapid and efficient acetonitrile (ACN)- and ultrasound-assisted on-bead trypsin digestion method combined with HPLC-MS/MS for the quantification of abrin isoforms in complex matrices. Specific peptides of abrin isoforms were generated by direct ACN-assisted trypsin digestion and analyzed by HPLC-HRMS. Combined with in silico digestion and BLASTp database search, fifteen marker peptides were selected for differential detection of abrin isoforms. The abrin in milk and plasma was enriched by immunomagnetic beads prepared by biotinylated anti-abrin polyclonal antibodies conjugated to streptavidin magnetic beads. The ultrasound-assisted on-bead trypsin digestion method was carried out under the condition of 10% ACN as denaturant solvent, the entire digestion time was further shortened from 90 min to 30 min. The four peptides of T3Aa,b,c,d, T12Aa, T15Ab, and T9Ac,d were chosen as quantification for total abrin, abrin-a, abrin-b, and abrin-c/d, respectively. The absolute quantification of abrin and its isoforms was accomplished by isotope dilution with labeled AQUA peptides and analyzed by HPLC-MS/MS (MRM). The developed method was fully validated in milk and plasma matrices with quantification limits in the range of 1.0-9.4 ng/mL for the isoforms of abrin. Furthermore, the developed approach was applied for the characterization of abrin isoforms from various fractions from gel filtration separation of the seeds, and measurement of abrin in the samples of biotoxin exercises organized by the Organization for the Prohibition of Chemical Weapons (OPCW). This study provided a recommended method for the differential identification of abrin isoforms, which are easily applied in international laboratories to improve the capabilities for the analysis of biotoxin samples.

Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry

Background: Monoclonal antibodies (mAbs) against cancer biomarkers are key reagents in diagnosis and therapy. One such relevant biomarker is a preferentially expressed antigen in melanoma (PRAME) that is selectively expressed in many tumors. Knowing mAb’s epitope is of utmost importance for understanding the potential activity and therapeutic prospective of the reagents. Methods: We generated a mAb against PRAME immunizing mice with PRAME fragment 161–415; the affinity of the antibody for the protein was evaluated by ELISA and SPR, and its ability to detect the protein in cells was probed by cytofluorimetry and Western blotting experiments. The antibody epitope was identified immobilizing the mAb on bio-layer interferometry (BLI) sensor chip, capturing protein fragments obtained following trypsin digestion and performing mass spectrometry analyses. Results: A mAb against PRAME with an affinity of 35 pM was obtained and characterized. Its epitope on PRAME was localized on residues 202–212, taking advantage of the low volumes and lack of fluidics underlying the BLI settings. Conclusions: The new anti-PRAME mAb recognizes the folded protein on the surface of cell membranes suggesting that the antibody’s epitope is well exposed. BLI sensor chips can be used to identify antibody epitopes.

Development of robust targeted proteomics assays for cerebrospinal fluid biomarkers in multiple sclerosis

Background Verification of cerebrospinal fluid (CSF) biomarkers for multiple sclerosis and other neurological diseases is a major challenge due to a large number of candidates, limited sample material availability, disease and biological heterogeneity, and the lack of standardized assays. Furthermore, verification studies are often based on a low number of proteins from a single discovery experiment in medium-sized cohorts, where antibodies and surrogate peptides may differ, thus only providing an indication of proteins affected by the disease and not revealing the bigger picture or concluding on the validity of the markers. We here present a standard approach for locating promising biomarker candidates based on existing knowledge, resulting in high-quality assays covering the main biological processes affected by multiple sclerosis for comparable measurements over time. Methods Biomarker candidates were located in CSF-PR (proteomics.uib.no/csf-pr), and further filtered based on estimated concentration in CSF and biological function. Peptide surrogates for internal standards were selected according to relevant criteria, parallel reaction monitoring (PRM) assays created, and extensive assay quality testing performed, i.e. intra- and inter-day variation, trypsin digestion status over time, and whether the peptides were able to separate multiple sclerosis patients and controls. Results Assays were developed for 25 proteins, represented by 72 peptides selected according to relevant guidelines and available literature and tested for assay peptide suitability. Stability testing revealed 64 peptides with low intra- and inter-day variations, with 44 also being stably digested after 16 h of trypsin digestion, and 37 furthermore showing a significant difference between multiple sclerosis and controls, thereby confirming literature findings. Calibration curves and the linear area of measurement have, so far, been determined for 17 of these peptides. Conclusions We present 37 high-quality PRM assays across 21 CSF-proteins found to be affected by multiple sclerosis, along with a recommended workflow for future development of new assays. The assays can directly be used by others, thus enabling better comparison between studies. Finally, the assays can robustly and stably monitor biological processes in multiple sclerosis patients over time, thus potentially aiding in diagnosis and prognosis, and ultimately in treatment decisions.