Common Repository of FBS Proteins (cRFP) To Be Added to a Search Database for Mass Spectrometric Analysis of Cell Secretome.

We propose to use cRFP (common Repository of FBS Proteins) in the MS (mass spectrometry) raw data search of cell secretomes. cRFP is a small supplementary sequence list of highly abundant fetal bovine serum proteins added to the reference database in use. The aim behind using cRFP is to prevent the contaminant FBS proteins from being misidentified as other proteins in the reference database, just as we would use cRAP (common Repository of Adventitious Proteins) to prevent contaminant proteins present either by accident or through unavoidable contacts from being misidentified as other proteins. We expect it to be widely used in experiments where the proteins are obtained from serum-free media after thorough washing of the cells, or from a complex media such as SILAC, or from extracellular vesicles directly.

Antimicrobial function of SHßAP, a novel hemoglobin ß chain-related antimicrobial peptide, isolated from the liver of skipjack tuna, Katsuwonus pelamis.

A 2.3 kDa of antimicrobial peptide was purified from an acidified liver extract of skipjack tuna, Katsuwonus pelamis, by preparative acid-urea-polyacrylamide gel electrophoresis and C18 reversed-phase HPLC. A comparison of the amino acid sequence of the purified peptide with those of other known polypeptides revealed high homology with the C-terminus of hemoglobin ß-chain; thus, this peptide was designated as the Skipjack Hemoglobin ß chain-related Antimicrobial Peptide (SHßAP). SHßAP showed potent antimicrobial activity against Gram-positive bacteria, such as Bacillus subtilis, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 6.5-57.0 µg/mL), Gram-negative bacteria, such as Escherichia coli D31, Pseudomonas aeruginosa, Salmonella enterica, Shigella sonnei, and two Vibrio parahaemolyticus species (MECs, 2.0-19.0 µg/mL), and against Candida albicans (MEC; 12.0 µg/mL) without significant hemolytic activity. Antimicrobial activity of this peptide was heatstable and pH resistant but is sensitive to proteases and salt. SHßAP did not show membrane permeabilization and killing ability. The secondary structural prediction and the homology modeling expected that this peptide formed an amphipathic α-helical structure. This is the first report the purification of a novel antimicrobial peptide related to the C-terminus of hemoglobin ß-chain from marine fish.

Quantification of protein markers monitoring the pre-analytical effect of blood storage time before plasma isolation using 15 N metabolically labeled recombinant proteins.

In the hospital, blood samples are collected to monitor patients' health states, and thus various protein-based clinical methods have been developed. However, some proteins are found to change in abundances during the process of blood collection and storage. In order to account such pre-analytical effects, we performed liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) on 15 selected proteins in plasma samples prepared by varying storage time and temperature of whole blood prior to plasma isolation. Two cytosolic proteins, profilin-1 (PFN1) and thymosin beta-4 (TMSB4X), were absolutely quantified using 15 N-labeled recombinant proteins spiked externally. The other 13 proteins were quantified in a relative way compared with the two reference proteins. Triplicated LC-MRM-MS measurements showed that the median CV of MRM peak areas was 5.7%. The amounts of PFN1 and TMSB4X increased rapidly depending on the storage time between blood collection and plasma preparation. It indicates the leakage of cellular components into the plasma fraction. Relative quantification further revealed that five proteins including PFN1, S10A8, S10A9, S10A11, and TMSB4X showed significant difference (P < 0.05). We further monitored PFN1 and TMSB4X on 40 samples collected for protein diagnostics under a typical clinical study condition. Compared with the plasma samples prepared within a day, the level of both PFN1 and TMSB4X increased in the plasma samples prepared from the blood collected the day before and kept overnight at 4°C (0.51 to 3.11 µg/mL for PFN1 and 0.98 to 5.36 µg/mL for TMSB4X in average). Our result suggests an effort of assuring plasma quality for accurate protein-based diagnosis or biomarker discovery and validation.

Role of Epigenetic Regulation by the REST/CoREST/HDAC Corepressor Complex of Moderate NANOG Expression in Chicken Primordial Germ Cells.

Primordial germ cells (PGCs), the precursors of gametes, have regulatory mechanisms involving transcription factors and epigenetic modifications. The transcription factor NANOG is a key regulator of germ cell and embryonic stem cell characteristics. However, the epigenetic regulation of NANOG with a histone deacetylase (HDAC) complex in PGCs has not been studied. In this study, we investigated the epigenetic regulation, and in particular the histone acetylation, of NANOG in chicken PGCs. Intriguingly, although NANOG was highly activated in chicken PGCs, the upstream region of its promoter was moderately suppressed by histone deacetylation. HDAC inhibition induced histone H3 lysine 9 acetylation (H3K9ac) and derepressed NANOG expression. Furthermore, knockdown studies revealed that HDAC complex members, such as RE1-silencing transcription factor (REST) and REST corepressor 3 (RCOR3), are important epigenetic modulators of NANOG expression in chicken PGCs. We demonstrate that moderate regulation of NANOG by the REST/CoREST/HDAC complex might be crucial for maintaining the integrity of PGCs.