Comparison of secretome from osteoblasts derived from sclerotic versus non-sclerotic subchondral bone in OA: A pilot study - Fig 5

(A) Fib3-1, (B) Fib3-2 and (C) Fib3-3 levels found in osteoblasts cell culture supernatant. Data are means +/- SD from 4 patients for Fib3-1, 7 patients for Fib3-2 and 9 patients for Fib3-3. Comparisons of mean values were performed by paired t-test.</p

Gene expression by NSC or SC osteoblasts.

<p>(A) less secreted proteins (B) more secreted proteins. The mRNA copy numbers were normalized against the corresponding copy number of HPRT mRNA and results are showed as the SC relative to NSC gene expression (mean ± SEM of five independent experiments performed with bone biopsy coming from different donors). In each experiment, each experimental condition was performed in duplicate. P: comparisons of mean values between NSC and SC were performed by unpaired t-test. Dotted blue line in each gene represents the result obtained with MS/MS in secretome with the corresponding protein.</p

MS/MS proteomic analysis of osteoblasts secretome.

<p>MS/MS proteomic analysis of osteoblasts secretome.</p

OMD1 and OMD2 levels found in healthy and OA human sera.

<p>(A) OMD1, (B) OMD2. Data are presented as scatter dot plots with median from 22 patients in each group. Comparisons of values were performed by Mann Whitney test. (C) Spearman correlation between OMD1 and OMD2 in serum, n = 44.</p

OMD1 and OMD2 levels found in osteoblasts cell culture supernatant.

<p>Data are means +/- SEM from 10 patients. Comparisons of mean values were performed by unpaired t-test.</p

Patient characteristics.

Patient characteristics.</p

Estimation of abundance of NSC osteoblasts secreted proteins using high top 3.

<p>Estimation of abundance of NSC osteoblasts secreted proteins using high top 3.</p

Western-blotting detection of osteomodulin using goat polyclonal antisera raised against entire osteomodulin.

<p>(A)(B) rhOMD: human recombinant osteomodulin (positive control), DMEM: concentration unconditioned culture supernatant (negative control), NSC: non sclerotic osteoblasts supernatant (N36, N38, N40), SC: sclerotic osteoblasts supernatant (S36, S38, S40), C1-6: six healthy patients albumin/IgG depleted serum, OA1-6: six OA patients before total knee replacement surgery albumin/IgG depleted serum. (C) Quantification of osteomodulin western blot performed on osteoblasts culture supernatants and on albumin/IgG depleted serum of 6 healthy patients (controls) and 6 patients before total knee replacement (OA). Quantification was made using ImageJ on total band and normalized with total protein charged visualized with reverse total protein stain (Pierce).</p

An Analytical Pipeline for MALDI In-Source Decay Mass Spectrometry Imaging

In-source decay (ISD) fragmentation as combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry allows protein sequencing directly from mass spectra. Acquisition of MALDI-ISD mass spectra from tissue samples is achieved using an appropriate MALDI matrix, such as 1,5-diaminonaphthalene (DAN). Recent efforts have focused on combining MALDI-ISD with mass spectrometry imaging (MSI) to provide simultaneous sequencing and localization of proteins over a thin tissue surface. Successfully coupling these approaches requires the development of new data analysis tools, but first, investigating the properties of MALDI-ISD as applied to mixtures of protein standards reveals a high sensitivity to the relative protein ionization efficiency. This finding translates to the protein mixtures found in tissues and is used to inform the development of an analytical pipeline for data analysis in MALDI-ISD MS imaging, including software to identify the most pertinent spectra, to sequence protein mixtures, and to generate ion images for comparison with tissue morphology. The ability to simultaneously identify and localize proteins is demonstrated by using the analytical pipeline on three tissue sections from porcine eye lens, resulting in localizations for crystallins and cytochrome c. The variety of protein identifications provided by MALDI-ISD-MSI between tissue sections creates a discovery tool, and the analytical pipeline makes this process more efficient

Venn diagrams of proteins with at least 1.5 fold expression change (p<0.05, student <i>t</i>-test) in reproductive organs of <i>Lymnaea</i><i>stagnalis</i> exposed to testosterone (T), cyproterone acetate (CPA), tributyltin and chlordecone during 21 days compared to (A) water controls and (B) solvent controls.

<p>Venn diagrams of proteins with at least 1.5 fold expression change (p<0.05, student <i>t</i>-test) in reproductive organs of <i>Lymnaea</i><i>stagnalis</i> exposed to testosterone (T), cyproterone acetate (CPA), tributyltin and chlordecone during 21 days compared to (A) water controls and (B) solvent controls.</p