Comprehensive Proteome Profiling of Platelet Identified a Protein Profile Predictive of Responses to An Antiplatelet Agent Sarpogrelate

Sarpogrelate is an antiplatelet agent widely used to treat arterial occlusive diseases. Evaluation of platelet aggregation is essential to monitor therapeutic effects of sarpogrelate. Currently, no molecular signatures are available to evaluate platelet aggregation. Here, we performed comprehensive proteome profiling of platelets collected from 18 subjects before and after sarpogrelate administration using LC-MS/MS analysis coupled with extensive fractionation. Of 5423 proteins detected, we identified 499 proteins affected by sarpogrelate and found that they strongly represented cellular processes related to platelet activation and aggregation, including cell activation, coagulation, and vesicle-mediated transports. Based on the network model of the proteins involved in these processes, we selected three proteins (cut-like homeobox 1; coagulation factor XIII, B polypeptide; and peptidylprolyl isomerase D) that reflect the platelet aggregation-related processes after confirming their alterations by sarpogrelate in independent samples using Western blotting. Our proteomic approach provided a protein profile predictive of therapeutic effects of sarpogrelate. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.1

Multiple-Cycle Polymeric Extracellular Vesicle Precipitation and Its Evaluation by Targeted Mass Spectrometry

The multiple roles of extracellular vesicles (EVs) in pathogenesis have received much attention, as they are valuable as diagnostic and prognostic biomarkers. We employed polymeric EV precipitation to isolate EVs from clinical specimens to overcome the limitations of ultracentrifugation (UC), such as low protein yields, a large volume of specimens used, and time requirements. Multiple-cycle polymeric EV precipitation was applied to enhance the purity of the EV fractions with a small sample volume. Then, the purity was assessed using a multiple reaction monitoring (MRM) panel consisting of alpha-2-macroglobulin (A2M), thrombospondin 1 (THBS 1), galectin 3 binding protein (LGALS3BP), and serum albumin (ALB). For purity evaluation, the EV fractions isolated from blood specimens were subjected to shotgun proteomics and MRM-based targeted proteomics analyses. We demonstrate that the modified method is an easy and convenient method compared with UC. In the shotgun proteomics analysis, the proteome profile of EV fraction contains 89% EV-related proteins by matching with the EVpedia database. In conclusion, we suggest that multiple-cycle polymeric EV precipitation is not only a more effective method for EV isolation for further proteomics-based experiments, but may also be useful for further clinical applications due to the higher EV yield and low sample requirements

Proteomic Analysis of the Meniscus Cartilage in Osteoarthritis

The distribution of differential extracellular matrix (ECM) in the lateral and medial menisci can contribute to knee instability, and changes in the meniscus tissue can lead to joint disease. Thus, deep proteomic identification of the lateral and medial meniscus cartilage is expected to provide important information for treatment and diagnosis of various knee joint diseases. We investigated the proteomic profiles of 12 lateral/medial meniscus pairs obtained from excess tissue of osteoarthritis patients who underwent knee arthroscopy surgery using mass spectrometry-based techniques and measured 75 ECM protein levels in the lesions using a multiple reaction monitoring (MRM) assay we developed. A total of 906 meniscus proteins with a 1% false discovery rate (FDR) was identified through a tandem mass tag (TMT) analysis showing that the lateral and medial menisci had similar protein expression profiles. A total of 131 ECM-related proteins was included in meniscus tissues such as collagen, fibronectin, and laminin. Our data showed that 14 ECM protein levels were differentially expressed in lateral and medial lesions (p < 0.05). We present the proteomic characterization of meniscal tissue with mass spectrometry-based comparative proteomic analysis and developed an MRM-based assay of ECM proteins correlated with tissue regeneration. The mass spectrometry dataset has been deposited to the MassIVE repository with the dataset identifier MSV000087753

The complete mitochondrial genome of the Korean endemic species Cobitis hankugensis (Kim, Park, Son & Nalbant, 2003)

Artemia has been considered as one of the most important live diets for crustacean and finfish larviculture as well as broodstocks. However, the basal nutrient of Artemia has been reported to be poor in polyunsaturated fatty acids (PUFA’s) especially eicosapentaenoic acids (EPA) and docosahexaenoic acids (DHA), essential fatty acids for larval normal growth and gonad maturity in shrimp broodstocks. Thus, the present study aimed at investigating the effect of different microalgal diets on fatty acid content, growth performances and survival rate of Artemia francisciana. The study was performed by culturing instar I nauplii of A. franciscana for 12 days at a stocking density of 100 nauplii/L and fed with one of these microalgae: Chaetoceros calcitrans (T1), Dunaliella salina (T2), Tetraselmis chuii (T3), and Nanochloropsis oculata (T4). The results showed that the different microalgal diets affected fatty acid content, growth and survival rate of A. fransicana. The highest DHA content was obtained from those Artemia fed on D. salina, p0.05. Another result indicated that EPA contents in the Artemia biomass were not significantly affected by the microalgal diets, p>0.05. In terms of growth and survival rate, A. franciscana fed on C. calcitrans and T. chuii had better growth and survival rate compared to that of Artemia fed on either D. salina or N. oculata, p<0.05. Due to the faster growth, it was also observed that Artemia fed on T. chuii started producing eggs on day 12. Further studies by feeding the Artemia with a mix of microalgal species either a mix of T. chuii and D. salina or a mix of C. calcitrans and D. salina are highly recommended for better PUFA contents, specific growth rate (SGR) as well as survival rates of Artemia

The complete mitochondrial genome of the Korean endemic species Cobitis hankugensis (Kim, Park, Son & Nalbant, 2003)

As one of efforts to conserve a genetic resource of the endemic cobitid species in the Korean peninsula, the complete mitogenome of Cobitis hankugensis (Kim, Park, Son & Nalbant, 2003) was determined using Illumina MiSeq system. The circular mitogenome was 16,557 bp length and encoded 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 tRNA genes, and a control region. Only the COX1 gene was identified with an aberrant initiation codon GTG, and an incomplete termination codon (T—/TA–) was identified in six PCGs including COX2, COX3, ND2, ND3, ND4, and Cytb genes. Phylogenetic analysis using 30 mitochondrial genomes belonging to Cobitidae, Botiidae, and Gyrinocheilidae showed that the highest identity (92.38%) with Kichulchoia brevifasciata (NC_027166). The complete mitogenome of C. hankugensis, an endemic species in Korea, will provide fundamental data on the evolutionary relationship of Cobitidae species