Protocol improvement and multisite validation of a digital soft agar colony formation assay for tumorigenic transformed cells intermingled in cell therapy products.

BACKGROUND AIMS: The administration of human cell-processed therapeutic products (hCTPs) is associated with a risk of tumorigenesis due to the transformed cellular contaminants. To mitigate this risk, these impurities should be detected using sensitive and validated assays. The digital soft agar colony formation (D-SAC) assay is an ultrasensitive in vitro test for detecting tumorigenic transformed cells in hCTPs. METHODS: In this study, we first evaluated the colony formation efficiency (CFE) precision of tumorigenic reference cells in positive control samples according to a previously reported D-SAC assay protocol (Protocol I) from multiple laboratories. However, the CFE varied widely among laboratories. Thus, we improved and optimized the test protocol as Protocol II to reduce variability in the CFE of tumorigenic reference cells. Subsequently, the improved protocol was validated at multiple sites. Human mesenchymal stromal cells (hMSCs) were used as model cells, and positive control samples were prepared by spiking them with HeLa cells. RESULTS: Based on the previously reported protocol, the CFE was estimated using an ultra-low concentration (0.0001%) of positive control samples in multiple plates. Next, we improved the protocol to reduce the CFE variability. Based on the CFE results, we estimated the sample size as the number of wells (Protocol II) and assessed the detectability of 0.0001% HeLa cells in hMSCs to validate the protocol at multiple sites. Using Protocol I yielded low CFEs (mean: 30%) and high variability between laboratories (reproducibility coefficient of variance [CV]: 72%). In contrast, Protocol II, which incorporated a relatively high concentration (0.002%) of HeLa cells in the positive control samples, resulted in higher CFE values (mean: 63%) and lower variability (reproducibility CV: 18%). Moreover, the sample sizes for testing were estimated as the number of wells per laboratory (314-570 wells) based on the laboratory-specific CFE (42-76%). Under these conditions, all laboratories achieved a detection limit of 0.0001% HeLa cells in hMSCs in a predetermined number of wells. Moreover, colony formation was not observed in the wells seeded with hMSCs alone. CONCLUSIONS: The D-SAC assay is a highly sensitive and robust test for detecting malignant cells as impurities in hCTPs. In addition, optimal assay conditions were established to test tumorigenic impurities in hCTPs with high sensitivity and an arbitrary false negative rate.

Characterization of the venom of the vermivorous cone snail Conus fulgetrum.

Over 200 components with molecular mass ranging mainly from 400 to 4000 Da were characterized from the venom of the vermivorous cone snail Conus fulgetrum that inhabit Egyptian Red Sea. One major component having a molecular mass of 2946 Da was purified by HPLC, and its primary structure was determined by a combination of Edman degradation and MS/MS analysis.

Identification of an antiviral component from the venom of the scorpion Liocheles australasiae using transcriptomic and mass spectrometric analyses.

Scorpion venom contains a variety of biologically active peptides. Among them, neurotoxins are major components in the venom, but it also contains peptides that show antimicrobial activity. Previously, we identified three insecticidal peptides from the venom of the Liocheles australasiae scorpion, but activities and structures of other venom components remained unknown. In this study, we performed a transcriptome analysis of the venom gland of the scorpion L. australasiae to gain a comprehensive understanding of its venom components. The result shows that potassium channel toxin-like peptides were the most diverse, whereas only a limited number of sodium channel toxin-like peptides were observed. In addition to these neurotoxin-like peptides, many non-disulfide-bridged peptides were identified, suggesting that these components have some critical roles in the L. australasiae venom. In this study, we also isolated a component with antiviral activity against hepatitis C virus using a bioassay-guided fractionation approach. By integrating mass spectrometric and transcriptomic data, we successfully identified LaPLA2-1 as an anti-HCV component. LaPLA2-1 is a phospholipase A2 having a heterodimeric structure that is N-glycosylated at the N-terminal region. Since the antiviral activity of LaPLA2-1 was inhibited by a PLA2 inhibitor, the enzymatic activity of LaPLA2-1 is likely to be involved in its antiviral activity.

A Facile Method for Preferential Modification of the N-Terminal Amino Group of Peptides Using Triazine-Based Coupling Reagents.

It has been shown that chemical modification of the peptide N-terminus with a charged tag greatly affects the fragmentation process caused by collision-induced dissociation to obtain more interpretable product ion spectra. In this study, we examined the selective introduction of a charged tag, 4-(guanidinomethyl)benzoic acid (Gmb), into the peptide N-terminus. After optimization of the reaction conditions, we found that the most effective conversion in terms of the reaction rate and selectivity was achieved by reacting the peptide with the active ester of Gmb, prepared using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) at pH 7. This method is applicable to the introduction of various carboxylic acid-containing compounds into the N-terminus of peptides, which will be useful not only for improvement of MS/MS fragmentation but also for various biochemical studies of peptides and proteins.

Complete de novo sequencing of antimicrobial peptides in the venom of the scorpion Isometrus maculatus.

Scorpion venom contains antimicrobial peptides (AMPs) in addition to neurotoxic peptides. Recent extensive transcriptomic analysis of venom glands of various scorpion species dramatically increased the number of identified AMPs. However, identification of peptides from genetic information requires reference sequences of similar peptides, and moreover, it is often difficult to predict post-translational modifications. In this study, we searched for unknown AMPs contained in the Isometrus maculatus scorpion venom based on the structural features, such as the hydrophobic nature and the lack of disulfide bonds, which are commonly observed in the majority of scorpion AMPs. Their primary structures were determined only by a de novo sequencing method using two types of MS instruments, which induce peptide fragmentation in a different fashion. Chemical derivatization techniques were also used to facilitate Leu/Ile discrimination. As a result, total 15 AMP candidates were discovered from the I. maculatus venom. Among them, three peptides were identified as AMPs by evaluating their biological activity. Other 12 candidate peptides were structurally related to the identified AMPs, possibly generated by enzymatic cleavage of the mature peptides in the venom.

N-Terminal Derivatization with Structures Having High Proton Affinity for Discrimination between Leu and Ile Residues in Peptides by High-Energy Collision-Induced Dissociation.

De novo sequencing is still essential in the identification of peptides and proteins from unexplored organisms whose sequence information is not available. One of the remaining problems in de novo sequencing is discrimination between Leu and Ile residues. The discrimination is possible based on differences in side chain fragmentation between Leu and Ile under high-energy collision-induced dissociation (HE-CID) conditions. However, this is observed only when basic residues, such as Arg and Lys, are present near the N- or C-terminal end. It has been shown that the charge derivatization at the N-terminal end by a quarternary ammonium or phosphonium moiety facilitates the side chain fragmentation by HE-CID. However, the effective backbone fragmentation by low-energy CID (LE-CID) is often hampered in those derivatives with a fixed charge. Previously, we demonstrated that the N-terminal charge derivatization with the structures having high proton affinity induced the preferential formation of b-ions under LE-CID conditions, allowing straightforward interpretation of product ion spectra. In the present study, we further investigated whether the same derivatization approach is also effective for discrimination between Leu and Ile under HE-CID conditions. Consequently, the side chain fragmentation of Leu and Ile residues was most effectively enhanced by the N-terminal derivatization with 4-(guanidinomethyl)benzoic acid among the tested structures. This derivatization approach, which is compatible with both HE- and LE-CID analysis, offers a straightforward and unambiguous de novo peptide sequencing method.