Peptides eluted from HLA-B27 of human splenocytes and blood cells reveal a similar but partially different profile compared to in vitro grown cell lines

Comparison of amino acid compositions of peptides eluted from HLA-B27 molecules of healthy individuals and patients with ankylosing spondylitis

“…1). This finding is also certified by our previously published data [28] where the same procedure, including the same specific monoclonal antibody TG1, was used for analysis of peptides isolated from human B27 spleen cells. In this case we showed that relative content of Arg at P2 was 68%, which was in very good agreement with previously published data.…”

“…The mean value difference was not statistically significant due to a large variation in both groups. The obtained mean relative concentrations of Arg at P2 are in contrast to its calculated value from registry of 370 sequenced peptides eluted from HLA-B27 of various cell lines (95%) [22] and also from splenocytes (68%) [28]. Table 1 Comparison of mean values of selected amino acid relative concentrations (%) in peptides isolated from PBL of HLA-B27 healthy (HLT) and diseased (AS) individuals and PBL pooled samples (HLT-P, AS-P) in nine cycles of SPED pool sequencing AA AS1 AS2 AS3 AS4 AS5 AS-P1 AS-P2 Mean HLT1 HLT2 HLT3 HLT4 HLT5 HLT6 HLT7 HLT8 HLT-P1 HLT-P2 HLT-P3 Mean PI The group of AS patients has higher mean values of Glu and lower of Gln compared to HLT donors in all cycles of SPED pool sequencing.…”

“…Isolation of peptides from HLA-B27 molecules and MALDI TOF MS analysis were described [28]. In short, individual and pooled samples were shortly sonicated and mixed with ice-cold lysis buffer containing 1% Triton X-100 and standard mixture of protease inhibitors.…”

“…After centrifugation, supernatant was loaded on Sepharose CL-4B column and consecutively on an affinity column containing immobilized monoclonal antibody TG1 (IgG2a specific to HLA-B27) [21]. Eluted fractions containing peptides were HPLC purified and the purity of isolated peptides was checked by MALDI TOF MS analysis [28]. The isolated peptide mixtures were then covalently attached to Sequelon-AA membrane using carbodiimide coupling according to manufacturer's protocol (Sequelon-AA Reagent kit, Millipore).…”

“…Software 610A was used to calibrate the instrument, quantify the PTH amino acid and compare the chromatograms. Mostly only one half of the membrane was processed [28].…”