Post-traumatic joint contracture is a debilitating consequence of trauma or surgical procedures. It is associated with fibrosis that develops regardless of the nature of initial trauma and results from complex biological processes associated with inflammation and cell activation. These processes accelerate production of structural elements of the extracellular matrix, particularly collagen fibrils. Although the increased production of collagenous proteins has been demonstrated in tissues of contracted joints, researchers have not yet determined the complex protein machinery needed for the biosynthesis of collagen molecules and for their assembly into fibrils. Consequently, the purpose of our study was to investigate key enzymes and protein chaperones needed to produce collagen-rich deposits. Using a rabbit model of joint contracture, our biochemical and histological assays indicated changes in the expression patterns of heat shock protein 47 and the a-subunit of prolyl 4-hydroxylase, key proteins in processing nascent collagen chains. Moreover, our study shows that the abnormal organization of collagen fibrils in the posterior capsules of injured knees, rather than excessive formation of fibril-stabilizing cross-links, may be a key reason for observed changes in the mechanical characteristics of injured joints. This result sheds new light on pathomechanisms of joint contraction, and identifies potentially attractive anti-fibrotic targets. ß

In this work, we pioneered a combination of ultralow flow (ULF) high-efficiency ultranarrow bore monolithic LC columns coupled to MS via a high-field asymmetric waveform ion mobility spectrometry (FAIMS) interface to evaluate the potential applicability for high sensitivity, robust, and reproducible proteomic profiling of low nanogram-level complex biological samples. As a result, ULF LC-FAIMS-MS brought unprecedented sensitivity levels and high reproducibility in bottom-up proteomic profiling. In addition, FAIMS improved the dynamic range, signal-to-noise ratios, and detection limits in ULF LC–MS-based measurements by significantly reducing chemical noise in comparison to the conventional nanoESI interface used with the same ULF LC–MS setup. Two, three, or four compensation voltages separated by at least 15 V were tested within a single LC–MS run using the FAIMS interface. The optimized ULF LC-ESI-FAIMS-MS/MS conditions resulted in identification of 2,348 ± 42 protein groups, 10,062 ± 285 peptide groups, and 15,734 ± 350 peptide-spectrum matches for 1 ng of a HeLa digest, using a 1 h gradient at the flow rate of 12 nL/min, which represents an increase by 38%, 91%, and 131% in respective identifications, as compared to the control experiment (without FAIMS). To evaluate the practical utility of the ULF LC-ESI-FAIMS-MS platform in proteomic profiling of limited samples, approximately 100, 1,000, and 10,000 U937 myeloid leukemia cells were processed, and a one-tenth of each sample was analyzed. Using the optimized conditions, we were able to reliably identify 251 ± 54, 1,135 ± 80, and 2,234 ± 25 protein groups from injected aliquots corresponding to ∼10, 100, and 1,000 processed cells.

Post-traumatic joint contracture is a frequent orthopaedic complication that limits the movement of injured joints, thereby severely impairing affected patients. Non-surgical and surgical treatments for joint contracture often fail to improve the range of motion. In this study, we tested a hypothesis that limiting the formation of collagen-rich tissue in the capsules of injured joints would reduce the consequences of the fibrotic response and improve joint mobility. We targeted the formation of collagen fibrils, the main component of fibrotic deposits formed within the tissues of injured joints, by employing a relevant rabbit model to test the utility of a custom-engineered antibody. The antibody was delivered directly to the cavities of injured knees in order to block the formation of collagen fibrils produced in response to injury. In comparison to the non-treated control, mechanical tests of the antibody-treated knees demonstrated a significant reduction of flexion contracture. Detailed microscopic and biochemical studies verified that this reduction resulted from the antibody-mediated blocking of the assembly of collagen fibrils. These findings indicate that extracellular processes associated with excessive formation of fibrotic tissue represent a valid target for limiting post-traumatic joint stiffness. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1038-1046, 2017.

Inhibition of the extracellular process of collagen fibril formation represents a new approach to limiting posttraumatic or postsurgical localized fibrosis. It has been demonstrated that employing a monoclonal antibody that targets the C-terminal telopeptide of the α2 chain of collagen I blocks critical collagen I-collagen I interaction, thereby reducing the amount of collagen deposits in vitro and in animal models. Here, we developed a chimeric variant of a prototypic inhibitory antibody of mouse origin. The structure of this novel antibody was analyzed by biochemical and biophysical methods. Moreover, detailed biochemical and biological studies were employed to test its antigen-binding characteristics. The ability of the chimeric variant to block formation of collagen fibrils was tested in vitro and in high-density cultures representing fibrotic processes occurring in the skin, tendon, joint capsule, and gingiva. The potential toxicity of the novel chimeric antibody was analyzed through its impact on the viability and proliferation of various cells and by testing its tissue cross-reactivity in sets of arrays of human and mouse tissues. Results of the presented studies indicate that engineered antibody-based blocker of localized fibrosis is characterized by the following: (i) a correct IgG-like structure, (ii) a high-affinity and high-specificity for a defined epitope, (iii) a great potential to limit accumulation of collagen-rich deposits, and (iv) a lack of cytotoxicity and non-specific tissue reactivity. Together, the presented study shows the great potential of the novel chimeric antibody to limit localized fibrosis, thereby setting ground for critical preclinical tests in a relevant animal model.

In this work, we developed an ultra-sensitive CE-MS/ MS method for bottom-up proteomics analysis of limited samples, down to sub-nanogram levels of total protein. Analysis of 880 and 88 pg of the HeLa protein digest standard by CE-MS/MS yielded ∼1100 ± 46 and ∼160 ± 59 proteins, respectively, demonstrating higher protein and peptide identifications than the current state-ofthe-art CE-MS/MS-based proteomic analyses with similar amounts of sample. To demonstrate potential applications of our ultrasensitive CE-MS/MS method for the analysis of limited biological samples, we digested 500 and 1000 HeLa cells using a miniaturized in-solution digestion workflow. From 1-, 5-, and 10-cell equivalents injected from the resulted digests, we identified 744 ± 127, 1139 ± 24, and 1271 ± 6 proteins and 3353 ± 719, 5709 ± 513, and 8527 ± 114 peptide groups, respectively. Furthermore, we performed a comparative assessment of CE-MS/MS and two reversed-phased nano-liquid chromatography (RP-nLC-MS/MS) methods (monolithic and packed columns) for the analysis of a ∼10 ng HeLa protein digest standard. Our results demonstrate complementarity in the protein-and especially peptide-level identifications of the evaluated CE-MS-and RP-nLC-MS-based methods. The techniques were further assessed to detect post-translational modifications and highlight the strengths of the CE-MS/ MS approach in identifying potentially important and biologically relevant modified peptides. With a migration window of ∼60 min, CE-MS/MS identified ∼2000 ± 53 proteins on average from a single injection of ∼8.8 ng of the HeLa protein digest standard. Additionally, an average of 232 ± 10 phosphopeptides and 377 ± 14 N-terminal acetylated peptides were identified in CE-MS/MS analyses at this sample amount, corresponding to 2-and 1.5-fold more identifications for each respective modification found by nLC-MS/MS methods.