Negative Electron Transfer Collision-Induced Dissociation of G-Quadruplexes: Uncovering the Guanine Radical Anion Loss Pathway.

G-quadruplex (G4) DNA can form highly stable secondary structures in the presence of metal cations, and research has shown its potential as a transcriptional regulator for oncogenes in the human genome. In order to explore the interactions of DNA with metal cations using mass spectrometry, employing complementary fragmentation methods can enhance structural information. This study explores the use of ion-ion reactions for sequential negative electron transfer collision-induced dissociation (nET-CID) as a complement to traditional ion-trap CID (IT-CID). The resulting nET-CID data for G4 anions with and without metal cations show an increase in fragment ion type diversity and yield of structurally informative ions relative to IT-CID. The nET-CID yields greater sequence coverage by virtue of fragmentation at the 3'-side of thymine residues, which is lacking with IT-CID. Potassium adductions to backbone fragments in IT-CID and nET-CID spectra were nearly identical. Of note is a prominent fragment resulting from a loss of a 149 Da anion seen in nET-CID of large, G-rich sequences, proposed to be radical anion guanine loss. Neutral loss of neutral guanine (151 Da) and deprotonated nucleobase loss (150 Da) have been previously reported, but this is the first report of radical anion guanine loss (149 Da). Confirmation of the identity of the 149 Da anion results from the examination of the homonucleobase sequence 5'-GGGGGGGG-3'. Loss of a charged adenine radical anion at much lower relative abundance was also noted for the sequence 5'-AAAAAAAA-3'. DFT modeling indicates that the loss of a nucleobase as a radical anion from odd-electron nucleic acid anions is a thermodynamically favorable fragmentation pathway for G.

Volume of preclinical xenograft tumors is more accurately assessed by ultrasound imaging than manual caliper measurements.

OBJECTIVE: The volume of subcutaneous xenograft tumors is an important metric of disease progression and response to therapy in preclinical drug development. Noninvasive imaging technologies suitable for measuring xenograft volume are increasingly available, yet manual calipers, which are susceptible to inaccuracy and bias, are routinely used. The goal of this study was to quantify and compare the accuracy, precision, and inter-rater variability of xenograft tumor volume assessment by caliper measurements and ultrasound imaging. METHODS: Subcutaneous xenograft tumors derived from human colorectal cancer cell lines (DLD1 and SW620) were generated in athymic nude mice. Experienced independent reviewers segmented 3-dimensional ultrasound data sets and collected manual caliper measurements resulting in tumor volumes. Imaging- and caliper-derived volumes were compared with the tumor mass, the reference standard, determined after resection. Bias, precision, and inter-rater differences were estimated for each mouse among reviewers. Bootstrapping was used to estimate mean and confidence intervals of variance components, intraclass correlation coefficients (ICCs), and confidence intervals for each source of variation. RESULTS: The average deviation from the true volume and inter-rater differences were significantly lower for ultrasound volumes compared with caliper volumes (P = .0005 and .001, respectively). Reviewer ICCs for ultrasound and caliper measurements were similarly low (1%), yet caliper volume variance was 1.3-fold higher than for ultrasound. CONCLUSIONS: Ultrasound imaging more accurately, precisely, and reproducibly reflects xenograft tumor volume than caliper measurements. These data suggest that preclinical studies using the xenograft burden as a surrogate end point measured by ultrasound imaging require up to 30% fewer animals to reach statistical significance compared with analogous studies using caliper measurements.