An antibody-based biomarker discovery method by mass spectrometry sequencing of complementarity determining regions

Autoantibodies are increasingly used as biomarkers in the detection of autoimmune disorders and cancer. Disease specific antibodies are generally detected by their binding to specific antigens. As an alternative approach, we propose to identify specific complementarity determining regions (CDR) of IgG that relate to an autoimmune disorder or cancer instead of the specific antigen(s). In this manuscript, we tested the technical feasibility to detect and identify CDRs of specific antibodies by mass spectrometry. We used a commercial pooled IgG preparation as well as purified serum IgG fractions that were spiked with different amounts of a fully human monoclonal antibody (adalimumab). These samples were enzymatically digested and analyzed by nanoLC Orbitrap mass spectrometry. In these samples, we were able to identify peptides derived from the CDRs of adalimumab. These peptides could be detected at an amount of 110 attomole, 5 orders of magnitude lower than the total IgG concentration in these samples. Using higher energy collision induced dissociation (HCD) fragmentation and subsequent de novo sequencing, we could successfully identify 50% of the detectable CDR peptides of adalimumab. In addition, we demonstrated that an affinity purification with anti-dinitrophenol (DNP) monoclonal antibody enhanced anti-DNP derived CDR detection in a serum IgG background. In conclusion, specific CDR peptides could be detected and sequenced at relatively low levels (attomole-femtomole range) which should allow the detection of clinically relevant CDR peptides in patient samples

Tissue proteomics outlines AGR2 AND LOX5 as markers for biochemical recurrence of prostate cancer

Although many patients are cured from prostate cancer (PCa) by surgery only, there are still patients who will experience rising prostate-specific antigen (PSA) levels after surgery, a condition known as biochemical recurrence (BCR). Novel protein prognostic markers in PCa tissue might enable finding better treatment for those patients experiencing BCR with a high chance of metastasis. In this study, we aimed to identify altered proteins in prostate cancer tissue, and to evaluate their potential role as prognostic markers. We used two proteomics strategies to analyse 34 prostate tumours (PCa) and 33 normal adjacent prostate (NAP) tissues. An independent cohort of 481 samples was used to evaluate the expression of three proteins: AGR2, FASN and LOX5 as prognostic markers of the disease. Tissue microarray immunohistochemical staining indicated that a low percentage of positive tumour cells for AGR2 (HR (95% CI) = 0.61 (0.43-0.93)), and a low percentage of positive tumour cells for LOX5 expression (HR (95% CI) = 2.53 (1.23-5.22)) are predictors of BCR after RP. In contrast, FASN expression had no prognostic value for PCa

TP53 mutated glioblastoma stem-like cell cultures are sensitive to dual mTORC1/2 inhibition while resistance in TP53 wild type cultures can be overcome by combined inhibition of mTORC1/2 and Bcl-2

Background: Glioblastoma is the most malignant tumor of the central nervous system and still lacks effective treatment. This study explores mutational biomarkers of 11 drugs targeting either the RTK/Ras/PI3K, the p53 or the Rb pathway using 25 patient-derived glioblastoma stem-like cell cultures (GSCs). Results: We found that TP53 mutated GSCs were approximately 3.5 fold more sensitive to dual inhibition of mammalian target of rapamycin complex 1 and 2 (mTORC1/2) compared to wild type GSCs. We identified that Bcl-2(Thr56/Ser70) phosphorylation contributed to the resistance of TP53 wild type GSCs against dual mTORC1/2 inhibition. The Bcl-2 inhibitor ABT-263 (navitoclax) increased sensitivity to the mTORC1/2 inhibitor AZD8055 in TP53 wild type GSCs, while sensitivity to AZD8055 in TP53 mutated GSCs remained unchanged. Conclusion: Our data suggest that Bcl-2 confers resistance to mTORC1/2 inhibitors in TP53 wild type GSCs and that combined inhibition of both mTORC1/2 and Bcl-2 is worthwhile to explore further in TP53 wild type glioblastomas, whereas in TP53 mutated glioblastomas dual mTORC1/2 inhibitors should be explored

Estimation of thermodynamic and physicochemical properties of the alkali astatides:On the bond strength of molecular astatine (At₂) and the hydration enthalpy of astatide (At⁻)

The recent accurate and precise determination of the electron affinity (EA) of the astatine atom At0 warrants a re-investigation of the estimated thermodynamic properties of At0 and astatine containing molecules as this EA was found to be much lower (by 0.4 eV) than previous estimated values. In this contribution we estimate, from available data sources, the following thermodynamic and physicochemical properties of the alkali astatides (MAt, M = Li, Na, K, Rb, Cs): their solid and gaseous heats of formation, lattice and gas-phase binding enthalpies, sublimation energies and melting temperatures. Gas-phase charge-transfer dissociation energies for the alkali astatides (the energy requirement for M+At− ➔ M0 + At0) have been obtained and are compared with those for the other alkali halides. Use of Born-Haber cycles together with the new AE (At0) value allows the re-evaluation of ΔHf (At0)g (=56 ± 5 kJ/mol); it is concluded that (At2)g is a weakly bonded species (bond strength &lt;50 kJ/mol), significantly weaker bonded than previously estimated (116 kJ/mol) and much weaker bonded than I2 (148 kJ/mol), but in agreement with the finding from theory that spin-orbit coupling considerably reduces the bond strength in At2. The hydration enthalpy (ΔHaq) of At− is estimated to be −230 ± 2 kJ/mol (using ΔHaq[H+] = −1150.1 kJ/mol), in good agreement with molecular dynamics calculations. Arguments are presented that the largest alkali halide, CsAt, like the smallest, LiF, will be only sparingly soluble in water, following the generalization from hard/soft acid/base principles that “small likes small” and “large likes large.”.</p