Peptide barcoding for one-pot evaluation of sequence–function relationships of nanobodies

Optimisation of protein binders relies on laborious screening processes. Investigation of sequence–function relationships of protein binders is particularly slow, since mutants are purified and evaluated individually. Here we developed peptide barcoding, a high-throughput approach for accurate investigation of sequence–function relationships of hundreds of protein binders at once. Our approach is based on combining the generation of a mutagenised nanobody library fused with unique peptide barcodes, the formation of nanobody–antigen complexes at different ratios, their fine fractionation by size-exclusion chromatography and quantification of peptide barcodes by targeted proteomics. Applying peptide barcoding to an anti-GFP nanobody as a model, we successfully identified residues important for the binding affinity of anti-GFP nanobody at once. Peptide barcoding discriminated subtle changes in KD at the order of nM to sub-nM. Therefore, peptide barcoding is a powerful tool for engineering protein binders, enabling reliable one-pot evaluation of sequence–function relationships.

Serum Level of Transferrin Unique Peptide Is Decreased in Patients With Acute Ischemic Stroke

Objectives: Free irons are transported into brain tissues by transferrin and play an important role in neuronal/glial cell damage. Lower serum levels of transferrin have been found in patients with ischemic stroke, compared with healthy subjects. In present study, we investigated whether transferrin unique peptide (TF-UP) could be employed as a serum biomarker for brain tissue damage in acute ischemic stroke.Methods: The venous blood samples of 94 ischemic stroke patients and 35 brain tumor-stroke mimics (BT-SM) patients were collected within the first 72 h (Median time 23.25, Interquartile range 60.75) of acute onset in the emergency room. Total TF-UP and total albumin unique peptide (Alb-UP) were identified with liquid chromatography/mass spectrometry (LC–MS/MS) and quantified by multiple reaction monitoring (MRM) method using labeled reference peptide (LRP) for further analysis.Results: Median ratio of total TF-UP/LRP was 0.85 (Interquartile range, 0.21) in the brain tumor-stroke mimics (BT-SM) group, and 0.45 (0.14) in the ischemic stroke group; median Alb-UP/LRP ratio was 0.66 (0.16) in the BT-SM group, and 0.55 (0.20) in the ischemic stroke group. The overall trend from low to high levels was statistically significant for TF-UP/LRP (P < 0.0001), but not for Alb-UP/LRP (P = 0.1667). According to the receiver operating characteristic (ROC) curve, the area under the curve (AUC) was 0.9565 and the optimal cutoff value of serum TF-UP was 0.6317, which yielded a sensitivity of 91.49% and a specificity of 88.57%. The odds ratio (95% confidence intervals) of serum TF-UP/LRP was 83.31 (23.43, 296.22, P < 0.0001).Conclusions: Serum TF-UP/LRP level is decreased in patients with acute ischemic stroke in comparison with brain tumor, and it may serve as a serum biomarker for the neuronal/glial cell damage in cerebral infarction.

Conserved unique peptide patterns (CUPP) online platform: peptide-based functional annotation of carbohydrate active enzymes

The CUPP platform includes a web server for functional annotation and sub-grouping of carbohydrate active enzymes (CAZymes) based on a novel peptide-based similarity assessment algorithm, i.e. protein grouping according to Conserved Unique Peptide Patterns (CUPP). This online platform is open to all users and there is no login requirement. The web server allows the user to perform genome-based annotation of carbohydrate active enzymes to CAZy families, CAZy subfamilies, CUPP groups and EC numbers (function) via assessment of peptide-motifs by CUPP. The web server is intended for functional annotation assessment of the CAZy inventory of prokaryotic and eukaryotic organisms from genomic DNA (up to 30MB compressed) or directly from amino acid sequences (up to 10MB compressed). The custom query sequences are assessed using the CUPP annotation algorithm, and the outcome is displayed in interactive summary result pages of CAZymes. The results displayed allow for inspection of members of the individual CUPP groups and include information about experimentally characterized members. The web server and the other resources on the CUPP platform can be accessed from https://cupp.info.

Oligopeptides for Immunotherapy Approaches in Ovarian Cancer Treatment

Background: Anti-ovarian cancer vaccines based on minimal immune determinants uniquely expressed in ovarian cancer biomarkers appear to promise a high level of sensitivity and specificity for ovarian cancer immunodiagnostics, immunoprevention, and immunotherapy. Methods: Using the Pir Peptide Match program, three ovarian cancer biomarkers – namely, sperm surface protein Sp17, WAP four-disulfide core domain protein 2, and müllerian-inhibiting substance – were searched for unique peptide segments not shared with other human proteins. Then, the unique peptide segments were assembled to define oligopeptides potentially usable as synthetic ovarian cancer antigens. Results and Conclusion: This study describes a methodology for constructing ovarian cancer biomarkerderived oligopeptide constructs that might induce powerful, specific, and non-crossreactive immune responses against ovarian cancer.

A sulfonium tethered peptide ligand rapidly and selectively modifies protein cysteine in vicinity

A unique peptide stabilization method provides a tethered sulfonium that can rapidly and selectively modify protein cysteine in close vicinity.