Label-free LC-MS/MS shotgun proteomics to investigate the anti-inflammatory effect of rCC16

Abstract

Clara cell protein (CC16) is an anti-inflammatory protein, which is expressed in the airway epithelium. It is involved in the development of airway inflammatory diseases, including chronic obstructive pulmonary disease and asthma. However, the exact molecular mechanism underlying its anti-inflammatory action remains to be fully elucidated. The aim of the present study was to define the protein profiles of the anti-inflammatory effect of CC16 in lipopolysaccharide (LPS)-treated rat tracheal epithelial (RTE) cells using shotgun proteomics. Protein extracts were obtained from control RTE cells, RTE cells treated with LPS and RTE cells treated with LPS and recombinant CC16 (rCC16). Subsequent label-free quantification and bioinformatics analyses identified 12 proteins that were differentially expressed in the three treatment groups as a cluster of five distinct groups according to their molecular functions. Five of the twelve proteins were revealed to be associated with the cytoskeleton: Matrix metalloproteinase-9, myosin heavy chain 10, actin-related protein-3 homolog, elongation factor 1-α-1 (EF-1-α-1), and acidic ribosomal phosphoprotein P0. Five of the twelve proteins were associated with cellular proliferation: DNA-dependent protein kinase catalytic subunit, EF-1-α-1, tyrosine 3-monooxygenase, caspase recruitment domain (CARD) protein 12 and adenosylhomocysteinase (SAHH) 3. Three proteins were associated with gene regulation: EF-1-α-1, SAHH 3 and acidic ribosomal phosphoprotein P0. Three proteins were associated with inflammation: Tyrosine 3-monooxygenase, CARD protein 12 and statin-related protein. ATPase (H+-transporting, V1 subunit A, isoform 1) was revealed to be associated with energy metabolism, and uridine diphosphate glycosyltransferase 1 family polypeptide A8 with drug metabolism and detoxification. The identified proteins were further validated using reverse transcription-quantitative polymerase chain reaction. These protein profiles, and their interacting protein network, may facilitate the elucidation of the molecular mechanisms underlying the anti-inflammatory effects of CC16.

Proteome Investigation of Rat Lungs subjected to Ex Vivo Perfusion (EVLP)

“…However, the genome only provides theoretical information of the protein levels while the proteome describes its actual content, which ultimately determines the phenotype. For this reason, genomic/transcriptomic investigations are recently giving way to proteomic approaches for a deepened and a more precise characterization of biological processes related to different clinical phenotypes and for the molecular monitoring of markers involved in respiratory diseases, such as COPD [13,14], cystic fibrosis [15], rhinitis [16], and the effect of anti-IgE therapy [17].…”

Exploration of key factors in Gingival Crevicular fluids from patients undergoing Periodontally Accelerated Osteogenic Orthodontics (PAOO) using proteome analysis

“…Impaired the lacunocanalicular network, including changed fluid flow, would activate intracellular molecular signaling pathways within osteocytes, actuating the secretion of mediators that could regulate bone remodeling, so as to contribute to the regional acceleration of bone remodeling (RAP). Kumar et al analyzed ALP and AST in human saliva and crevicular fluid, and found that the enzyme activities of ALP and AST increased after corticotomy and reached a peak at the 6th week, and then decreased at the 3rd and 6th months [ 10 ]. Fernandes LSDMCP et al have shown that GCF during the movement of canines were quantified in a multiplex immunoassay, and differences on biomarkers expression occurred at specific time points (p < 0.05), but a distinct pattern was not observed [ 11 ].…”

“…Following the addition of 100 mmol/L DTT(DL-Dithiothreitol) to a final concentration of 10 mmol/L, the protein fractions were mixed at 56℃ for 60 min, and then diluted 10x with 250 mmol/L IAM(Iodoacetamide) and maintained in dark for 60 min. Finally, the samples were digested with trypsin (substrate to enzyme mass at a mass ratio of 50:1 at 37℃ for 12 h [ 10 ].…”

LC-MS/MS shotgun proteomics reveals biochemical mechanisms of Paspalum fasciculatum tolerance to Pb-stress

“…Mass spectrometry (MS)-based proteomic technologies are powerful tools used for large-scale protein identification and quantitation (Cravatt et al 2007). Label-free shotgun proteomics is a very effective technique for the identification of peptides, determining the abundance of the proteins, and subsequently, obtaining a global protein profile of a sample, which is important for applications in complex biological systems (Pang et al 2016, Han et al 2021, Shen et al 2021, Szuba et al 2021. Consequently, proteomics is a very useful technique to study plant responses to heavy metal stress.…”