Quantitative proteomics analysis of cartilage response to mechanical injury and cytokine treatment

Abstract

Mechanical damage at the time of joint injury and the ensuing inflammatory response associated with elevated levels of pro-inflammatory cytokines in the synovial fluid, are reported to contribute to the progression to osteoarthritis after injury. In this exploratory study, we used a targeted proteomics approach to follow the progression of matrix degradation in response to mechanical damage and cytokine treatment of human knee cartilage explants, and thereby to study potential molecular biomarkers. This proteomics approach allowed us to unambiguously identify and quantify multiple peptides and proteins in the cartilage medium and explants upon treatment with +/− injurious compression +/−cytokines, treatments that mimic the earliest events in post-traumatic OA. We followed degradation of different protein domains, e.g., G1/G2/G3 of aggrecan, by measuring representative peptides of matrix proteins released into the medium at 7 time points throughout the 21-day culture period. COMP neo epitopes, which were previously identified in the synovial fluid of knee injury/OA patients, were also released by these human cartilage explants treated with cyt and cyt+inj. The absence of collagen pro-peptides and elevated levels of specific COMP and COL3A1 neo-epitopes after human knee trauma may be relevant as potential biomarkers for post-traumatic OA. This model system thereby enables study of the kinetics of cartilage degradation and the identification of biomarkers within cartilage explants and those released to culture medium. Discovery proteomics revealed that candidate proteases were identified after specific treatment conditions, including MMP1, MMP-3, MMP-10 and MMP-13.

Analysis of “old” proteins unmasks dynamic gradient of cartilage turnover in human limbs

“…Discovery experiments (nontargeted MS) were performed on a quadrupole Orbitrap benchtop mass spectrometer (Q Exactive) (Thermo Fisher Scientific, Waltham, WA) equipped with an EASY-nLC 1000 system (Thermo Scientific, Waltham, MA) as previously described ( 55 ). An MS scan [400 to 1200 mass/charge ratio ( m / z )] was recorded in the Orbitrap mass analyzer set at a resolution of 70,000 at 200 m / z , 1 × 10 6 automatic gain control target, and 100-ms maximum ion injection time.…”

Proteomic comparison of osteoarthritic and reference human menisci using data-independent acquisition mass spectrometry

“…which is based on guanidine hydrochloride. This method is not optimal for collagen extraction and depending on the extent of collagen cross-linking or subtypes in different samples, the results may vary 32 . It might therefore be premature to make any interpretation about collagens from our study, without further validation.…”

Differential protein expression in human knee articular cartilage and medial meniscus using two different proteomic methods: a pilot analysis

“…Finally, the organic solvent concentration increased up to 90% in 5 min and kept at 90% for 7 min. The MS1 and MS2 scans were performed as previously described [ 14 ], with the exception of MS2 resolution that was set to 17,500 in this study. The system was controlled by Xcalibur™ Software (Thermo Fisher Scientific).…”

“…The raw DDA data were searched against the human Swiss-Prot database (Swiss-Prot_2015_06, containing 20,200 sequences) using Proteome Discoverer™ 2.1 Software (Thermo Fisher Scientific) as previously described [ 14 ], with the exceptions of included modifications, which in this study was: static modification: cysteine carbamidomethylation and dynamic modifications: N-terminal acetylation and methionine oxidation. In the data analysis, the top 3 peptides are averaged, which is based on a study that reported that the average MS signal response for the three most intense tryptic peptides signals per mole of protein is constant [ 15 ].…”