Optimized Glycopeptide Enrichment Method-It Is All About the Sauce.

Protein glycosylation is a family of posttranslational modifications that play a crucial role in many biological pathways and diseases. The enrichment and analysis of such a diverse family of modifications are very challenging because of the number of possible glycan-peptide combinations. Among the methods used for the enrichment of glycopeptides, boronic acid never lived up to its promise. While most studies focused on improving the affinity of the boronic acids to the sugars, we discovered that the buffer choice is just as important for successful enrichment if not more so. We show that an amine-less buffer allows for the best glycoproteomic coverage, in human plasma and brain specimens, improving total quantified glycopeptides by over 10-fold, and reaching 1598 N-linked glycopeptides in the brain and 737 in nondepleted plasma. We speculate that amines compete with the glycans for boronic acid binding, and therefore the elimination of them improved the method significantly.

CDR3 and V genes show distinct reconstitution patterns in T cell repertoire post-allogeneic bone marrow transplantation.

Restoration of T cell repertoire diversity after allogeneic bone marrow transplantation (allo-BMT) is crucial for immune recovery. T cell diversity is produced by rearrangements of germline gene segments (V (D) and J) of the T cell receptor (TCR) α and ß chains, and selection induced by binding of TCRs to MHC-peptide complexes. Multiple measures were proposed for this diversity. We here focus on the V-gene usage and the CDR3 sequences of the beta chain. We compared multiple T cell repertoires to follow T cell repertoire changes post-allo-BMT in HLA-matched related donor and recipient pairs. Our analyses of the differences between donor and recipient complementarity determining region 3 (CDR3) beta composition and V-gene profile show that the CDR3 sequence composition does not change during restoration, implying its dependence on the HLA typing. In contrast, V-gene usage followed a time-dependent pattern, initially following the donor profile and then shifting back to the recipients' profile. The final long-term repertoire was more similar to that of the recipient's original one than the donor's; some recipients converged within months, while others took multiple years. Based on the results of our analyses, we propose that donor-recipient V-gene distribution differences may serve as clinical biomarkers for monitoring immune recovery.

Prediction of Specific TCR-Peptide Binding From Large Dictionaries of TCR-Peptide Pairs.

Current sequencing methods allow for detailed samples of T cell receptors (TCR) repertoires. To determine from a repertoire whether its host had been exposed to a target, computational tools that predict TCR-epitope binding are required. Currents tools are based on conserved motifs and are applied to peptides with many known binding TCRs. We employ new Natural Language Processing (NLP) based methods to predict whether any TCR and peptide bind. We combined large-scale TCR-peptide dictionaries with deep learning methods to produce ERGO (pEptide tcR matchinG predictiOn), a highly specific and generic TCR-peptide binding predictor. A set of standard tests are defined for the performance of peptide-TCR binding, including the detection of TCRs binding to a given peptide/antigen, choosing among a set of candidate peptides for a given TCR and determining whether any pair of TCR-peptide bind. ERGO reaches similar results to state of the art methods in these tests even when not trained specifically for each test. The software implementation and data sets are available at https://github.com/louzounlab/ERGO. ERGO is also available through a webserver at: http://tcr.cs.biu.ac.il/.

New perspectives on the mutated NGLY1 enigma.

The enzyme N-glycanase 1 (NGLY1) is considered a component of the endoplasmic reticulum-associated degradation (ERAD) machinery and clinical manifestations of its dysfunction include global developmental delay, a movement disorder, peripheral neuropathy, liver disorders, microcephaly, diminished reflexes and seizures. Although several mutations in NGLY1 have been identified, the relation between the defected protein and the above described pathologies is yet unknown. We hypothesised that NGLY1 failure to degrade certain proteins may result in their accumulation and overexpression and used a systems biology approach to identify proteins that may be affected by NGLY1 deficiency. Genes that interact with the NGLY1 gene according to BioGRID database of physical and genetic interactions were analysed with STRING Protein-Protein interaction database. Network analysis identified FAF1 (Fas-Associated Factor 1), an apoptosis-potentiating protein, as a possible degradation substrate of NGLY1. Examination of normal tissue microarrays demonstrated that FAF1-to-NGLY1 ratio is maximal (more than 3:1) in skeletal muscle and brain tissues microarrays. This evidence may explain the pathologies in brain and muscle tissues of patients with mutated NGLY1. To test this hypothesis, laboratory studies that will assess if FAF1 protein is overexpressed in tissues of patients with mutated NGLY1 are required.