Bottom-up Histone Post-translational Modification Analysis using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Tandem Mass Spectrometry.

The amino acid position within a histone sequence and the chemical nature of post-translational modifications (PTMs) are essential for elucidating the "Histone Code". Previous work has shown that PTMs induce specific biological responses and are good candidates as biomarkers for diagnostics. Here, we evaluate the analytical advantages of trapped ion mobility (TIMS) with parallel accumulation-serial fragmentation (PASEF) and tandem mass spectrometry (MS/MS) for bottom-up proteomics of model cancer cells. The study also considered the use of nanoliquid chromatography (LC) and traditional methods: LC-TIMS-PASEF-ToF MS/MS vs nLC-TIMS-PASEF-ToF MS/MS vs nLC-MS/MS. The addition of TIMS and PASEF-MS/MS increased the number of detected peptides due to the added separation dimension. All three methods showed high reproducibility and low RSD in the MS domain (<5 ppm). While the LC, nLC and TIMS separations showed small RSD across samples, the accurate mobility (1/K0) measurements (<0.6% RSD) increased the confidence of peptide assignments. Trends were observed in the retention time and mobility concerning the number and type of PTMs (e.g., ac, me1-3) and their corresponding unmodified, propionylated peptide that aided in peptide assignment. Mobility separation permitted the annotation of coeluting structural and positional isomers and compared with nLC-MS/MS showed several advantages due to reduced chemical noise.

Evaluation of a Commercial TIMS-Q-TOF Platform for Native Mass Spectrometry.

Mass-spectrometry based assays in structural biology studies measure either intact or digested proteins. Typically, different mass spectrometers are dedicated for such measurements: those optimized for rapid analysis of peptides or those designed for high molecular weight analysis. A commercial trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) platform is widely utilized for proteomics and metabolomics, with ion mobility providing a separation dimension in addition to liquid chromatography. The ability to perform high-quality native mass spectrometry of protein complexes, however, remains largely uninvestigated. Here, we evaluate a commercial TIMS-Q-TOF platform for analyzing noncovalent protein complexes by utilizing the instrument's full range of ion mobility, MS, and MS/MS (both in-source activation and collision cell CID) capabilities. The TIMS analyzer is able to be tuned gently to yield collision cross sections of native-like complexes comparable to those previously reported on various instrument platforms. In-source activation and collision cell CID were robust for both small and large complexes. TIMS-CID was performed on protein complexes streptavidin (53 kDa), avidin (68 kDa), and cholera toxin B (CTB, 58 kDa). Complexes pyruvate kinase (237 kDa) and GroEL (801 kDa) were beyond the trapping capabilities of the commercial TIMS analyzer, but TOF mass spectra could be acquired. The presented results indicate that the commercial TIMS-Q-TOF platform can be used for both omics and native mass spectrometry applications; however, modifications to the commercial RF drivers for both the TIMS analyzer and quadrupole (currently limited to m/z 3000) are necessary to mobility analyze protein complexes greater than about 60 kDa.

Apical dose versus volume dose of Ruthenium-106 brachytherapy for uveal melanoma.

OBJECTIVE: Ruthenium-106 brachytherapy is commonly used to treat uveal melanomas. Most centres prescribe a radiation dose to the tumour apex that is calculated with the tumour located in the centre of the plaque. Recent work suggests that D99%-the minimum radiation dose delivered to 99% of tumour volume-may be a better predictor of tumour control than apex dose. Both dosing regimens may be affected by tumour and treatment variables differently. We explored the effect of differences in these variables on volume and apex dose using a 3-dimensional planning model. METHODS: The time required to deliver 100 Gy to the tumour apices of representative tumours ranging from 2- to 6-mm thickness with central plaque positioning was calculated in Plaque Simulator™. This treatment time was used for further calculations, including D99% with central plaque placement, and apical and tumour volume doses when tumour and plaque characteristics were altered, including eccentric plaque placement, either away from (tilt) or along (offset) scleral surface, tumour shape, and plaque type. RESULTS: D99% was always greater than the apex dose when plaques were placed centrally, and the difference increased with tumour thickness. Increasing degrees of tumour offset reduced apical dose and D99%, with a greater effect on apical dose for thicker and D99% for thinner tumours, respectively. Differences in tumour shape and plaque type had idiosyncratic effects on apical and volume dosing. CONCLUSION: D99% and apex dose are affected by tumour and treatment characteristics in different ways, highlighting the complexity of radiation delivery to uveal tumours.

Effect of 2-methoxyestradiol on mammary tumor initiation and progression.

BACKGROUND: The anti-cancer agent 2-methoxyestradiol (2-ME) has been shown to have anti-proliferative and anti-angiogenic properties. Previously, the effect of 2-ME on early- and late-stage breast cancer (BC) was investigated in vivo using a transgenic mouse model (FVB/N-Tg(MMTV-PyVT)) of spontaneous mammary carcinoma. Anti-tumor effects were observed in late-stage BC with no effect on early-stage BC. Given the contrasting results obtained from the different BC stages, we have now investigated the effect of 2-ME when administered before the appearance of palpable tumors. METHODS: Each mouse received 100 mg/kg 2-ME on day 30 after birth, twice per week for 28 days, while control mice received vehicle only. Animals were terminated on day 59. Lung and mammary tissue were obtained for immunohistochemical analysis of CD163 and CD3 expression, and histological examination was performed to analyze tumor necrosis. Additionally, blood samples were collected to measure plasma cytokine levels. RESULTS: 2-ME increased tumor mass when compared to the untreated animals (p = .0139). The pro-tumorigenic activity of 2-ME was accompanied by lower CD3+ T-cell numbers in the tumor microenvironment (TME) and high levels of the pro-inflammatory cytokine interleukin (IL)-1ß. Conversely, 2-ME-treatment resulted in fewer CD163+ cells detectable in the TME, increased levels of tumor necrosis, increased IL-10 plasma levels, and low IL-6 and IL-27 plasma levels. CONCLUSION: Taken together, these findings suggest that 2-ME promotes early-stage BC development.

Ameloblastic carcinoma: A systematic review.

BACKGROUND: Ameloblastic carcinoma (AC) is the most common odontogenic malignancy, constituting approximately 30% of cases in this category. Literature is sparse on malignant odontogenic neoplasms, with a large proportion of current knowledge derived from case reports or small case series. METHODS: A systematic review of case series/case reports of AC was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) Statement guidelines. Demographic and clinical information, including duration of the lesion, location, clinical presentation and radiologic features, were analysed. Additionally, the origin of the lesion (primary/secondary), Ki-67 proliferation index, treatment performed, metastasis, tumour recurrence and prognosis were collected for analysis. RESULTS: A total of 126 studies, including 285 individual cases of AC, were included in this review. Patients presented with a near-equal distribution of painless and painful swellings. ACs presented at a median age of 45 years, with a male-to-female ratio of 1:2. The mandible was most frequently involved, with rare cases extending to involve more than one region, including crossing the midline. Although most lesions presented with poorly-demarcated borders (52.6%), unilocular lesions with well-demarcated borders (47.4%) comprised a substantial number in the sample. The proliferation index was only reported in 27 cases, with a mean score of 42% and a wide range. The probability of tumour recurrence increased, and the survival probability decreased with prolonged follow-up duration. CONCLUSION: This study provides more comprehensive, up-to-date descriptive data on these rare odontogenic malignancies, aiding clinicians and Pathologists with the diagnosis and surgeons in their management of cases.

Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry.

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.

Quantifying the effect of eccentric ruthenium plaque placement on tumor volume dose.

Purpose: Ruthenium-106 brachytherapy is a common treatment for small to medium-sized uveal melanomas. In certain clinical contexts, plaques may be placed eccentrically to tumor center. The effect of plaque decentration, a common radiation dose measurement in radiotherapy: D98%, the percentage of the tumor volume receiving at least 98% of the prescribed dose (a commonly used term in radiation oncology), is unknown. We investigated this using two commonly used plaques (CCA and CCB; Eckert & Ziegler, BEBIG GmbH) in silico. Material and methods: Using a Plaque Simulator™ (Eye Physics) plaque modelling software, treatment time required to deliver 100 Gy D98% with central plaque placement was calculated for both plaque models, treating tumors with basal dimensions of 10 mm (CCB plaque only) and 7 mm (CCA and CCB plaques), and a range of thicknesses. D98% was calculated for plaque-tumor edge distances of 0-5 mm. Additionally, we defined minimum plaque-tumor edge distances, at which D98% fell by 10% and 5% (safety margins). Results: D98% decreased as plaque-tumor edge distance decreased, i.e. as plaque eccentricity increased. Minor (< 1 mm) plaque decentration caused minimal D98% changes across tumor thicknesses. Safety margins did not follow a consistent pattern. Conclusions: Eccentric plaque placement reduces the radiation dose delivered to choroidal tumors. Both tumor (thickness, diameter) and plaque (size, location) characteristics are important D98% modulators. Further investigation of the effect of these characteristics and dose to organs at risk is essential.