Also, lectin enrichment approach was employed to enrich glycoproteins utilizing a mix ture of three different lectins wheat germ agglutinin, concanavalin A and jacalin. These lectins have different binding specificities and thereby permit enrichment of a broader coverage of glycoproteins. The lectin enriched frac tions were subjected to SDS Web page and SCX fractionation. All of those fractions have been analyzed on a Fourier transform LTQ Orbitrap Velos mass spectrometer. The workflow il lustrating the measures involved in the proteomic evaluation of OA synovial fluid is shown in Figure 1. 124,380 peptide spectrum matches generated from the mass spectrometric analysis of 112 fractions of depleted and lectin enriched OA synovial fluid resulted in the iden tification of 5,544 peptides corresponding to 677 proteins.
The amount of proteins identified from the depleted and lectin enriched fractions are summarized in Added file 1A and 1B, respectively. In the 300 lectin enriched pro teins identified, 171 proteins were already recognized to be glycosylated from the data offered selleck chemicals EPZ005687 in Human Protein Reference Database. The complete list of all proteins and peptides identified in our study are pro vided in Extra files two and three, respectively. The relative abundance from the 25 most abundant proteins identified is supplied in More file 4. Classification determined by gene ontology annotation GO based annotation was made use of to categorize the pro teins determined by their subcellular localization, molecular function and biological processes.
Signal peptide and transmembrane domain analysis from the identified pro teins was done by using the domains motif details obtainable in HPRD. Out of 677 proteins, 400 proteins have been found to possess a signal peptide, 113 selelck kinase inhibitor have trans membrane domains and 77 proteins possessed each. Classification depending on the subcellular localization indicated that 40% of proteins have been extracel lular. Proteins were also localized to cytoplasm, plasma membrane and nucleus. According to their molecular function, proteins have been classi fied as constituents with the extracellular matrix or these involved in transporter activity, cell adhesion molecule activity, protease inhibitor activity and complement activity. Biological process based categorization showed that a majority of them played a function in cell communication and signaling, cell development and or maintenance, protein metabol ism and immune response. Proteins previously reported in OA synovial fluid Many proteins reported earlier in OA synovial fluid were identified in our study confirming the validity of your ex perimental strategy employed by us.