Supplementary MaterialsSupplementary figure 1: IceLogo plot of amino acid frequencies at the termini of DUCAF peptidome Frequencies of proteins are plotted in iceLogo against amino acid frequency in the human proteome for the N terminus (A) and C terminus (B). derived from two different cell types (dendritic cells Retigabine and EBV-transformed B cells) were identified with mass spectrometry and the binding core and N- and C-terminal residues of a total of 16,568 peptides were analysed using the frequencies of the amino acids in the human proteome. Similar binding motifs were found as well as comparable conservations in the N- and C-terminal residues. Furthermore, the terminal conservations of these ligandomes were compared to the N- and C-terminal conservations of the ligandome acquired from dendritic cells homozygous for HLA-DRB1*04:01. Again, comparable conservations were evident with only minor differences. Taken together, these data show that there are conservations in the terminal residues of peptides, presumably the result of the activity of proteases involved in antigen processing. Electronic supplementary material The online version of this article (10.1007/s00251-019-01129-6) contains supplementary material, which is available to authorized users. to remove nuclei and insoluble materials. Peptide elution and isolation from affinity-purified HLA-DR molecules A complete of 2.5?mg pan-HLA-DR antibody (B8.11.2) was coupled to 1-ml protein-A-Sepharose CL4B beads by dimethyl pimelimidate crosslinking (Schneider et al. 1982). Beads had been prewashed with lysis buffer by gravitation in little columns. DUCAF and DC lysates had been precleared using Sepharose CL4B (GE Health care) beads and HLA molecules had been isolated using 100?l of Ab-protA-Sepharose beads for every 100??106 cells. After isolation, the beads had been washed with lysis buffer accompanied by washing measures with low salt buffer (120?mM Retigabine NaCl, 20?mM Tris-HCl, pH?8.0), high salt buffer (1?M NaCl, 20?mM Tris-HCl, pH?8.0), zero salt buffer (20?mM Tris-HCl, pH?8.0) and low Tris buffer (10?mM Tris-HCl, pH?8.0). The peptides had been subsequently eluted with 10% acetic acid. Peptide identification by mass spectrometry Mass spectrometry (MS) evaluation of HLA-eluted peptides was performed as referred to previously (van Lummel et al. 2011) with some adjustments. After elution, HLA molecules and HLA-binding peptides had been separated by selective elution from a little C18 column (Oasis, Waters) in two fractions with 20% and 30% acetonitrile (Lecaille et al. 2002). DUCAF peptides had been pre-fractionated into 25 fractions using SCX HPLC. Subsequently, the HLA-peptides had been analysed via on-range C18-nano-HPLC-MS with something consisting Rabbit Polyclonal to SNIP of a straightforward nLC 1000 gradient HPLC program (Thermo, Bremen, Germany) and a Q-Exactive mass spectrometer (Thermo). Fractions had been injected onto a homemade precolumn (100?m??15?mm; Reprosil-Pur C18-AQ 3?m, Dr. Maisch, Ammerbuch, Germany) and eluted with a homemade analytical nano-HPLC column (15?cm??50?m; Reprosil-Pur C18-AQ 3 um). The gradient was operate from 0 Retigabine to 30% solvent B (10/90/0.1 drinking water/ACN/FA data source (67,211 entries) using Mascot Edition 2.2.04 (Matrix Technology) with the next configurations: 10?ppm and 20 millimass devices deviation for precursor and fragment masses, respectively; simply no enzyme was specified. In percolator, an FDR of 1% was arranged, with the excess condition of Retigabine a mascot rating of at least 35. The mass spectrometry proteomics data have already been deposited to the ProteomeXchange Consortium via the Satisfaction (Perez-Riverol et al. 2019) partner repository with the dataset identifier PXD014253 (Deutsch et al. 2017). Gibbs clustering The peptides had been clustered using the Gibbs Clustering device available on-line (Gibbs Cluster 1.1 server) (Andreatta and Lund 2013). The amount of clusters was arranged to 1C4 and motif size to 9 proteins. The rest of the configurations was utilized as default. http://www.cbs.dtu.dk/services/GibbsCluster/ NetMHCIIPan 3.2 The binding cores of the HLA-DR-derived ligandomes had been predicted using NetMHCIIPan 3.2 logarithm applying the default configurations (Jensen et al. 2018). The resulting binding motif was when compared to binding motif referred to in the SYFPEITHI data source (Rammensee et al. 1999). http://www.cbs.dtu.dk/services/NetMHCIIpan/ iceLogo Sequence logos were generated by plotting the amino acid sequences against a positive reference group of the proteome using iceLogo version 1.3.8 (Colaert et al. 2009). Statistical evaluation Statistical evaluation was performed with Stata SE 14.1 (StataCorp LLC). To check for significance, the Chi-square check was performed. The Bonferroni technique was utilized to improve for multiple tests for all statistical analyses (5 amino acid groups??3 pairwise comparisons??2 termini??4.