Purpose Mutations in the and (mutations can lead to high levels

Purpose Mutations in the and (mutations can lead to high levels of 2HG circulating in the blood and whether serum 2HG can be used as a biomarker for mutational status and tumor burden in ICC. 2HG. Conclusions This study indicates that circulating 2HG may be a surrogate biomarker of or mutation status in ICC and that circulating 2HG levels may correlate directly with tumor burden. and (mutations confined primarily to a small number of cancer types which is then further limited when considering a relatively rare malignancy such as ICC. We have previously identified elevated levels of 2HG in the tumor tissue of or mutation. Accurate detection and quantification of serum 2HG could potentially serve as an efficient and less-invasive method of assessing a patient’s response to IDH-targeted therapies once promising drugs currently undergoing preclinical evaluation enter into clinical testing (17 18 In this study we therefore sought to characterize serum 2HG levels as a biomarker of mutational status and its association with tumor burden in gene at nucleotide positions c.394 and c.395 (amino acid position p.R132) were identified using a multiplexed mutational profiling platform that has been previously described and clinically implemented (6 19 Rare mutations that have been reported in other tumor types were not evaluated (20). Sanger sequencing was used to identify mutations in the gene at exon 4 (including mutations at codons p.140 and p.172) using methods and polymerase chain reaction primers that have been previously reported (6). Labeled PCR products were separated using an ABI PRISM 3730 DNA Analyzer and the data were interpreted with GeneMapper Analysis Software (Life Technologies/Applied Biosystems). Circulating 2-Hydroxyglutarate analysis Serum was isolated from whole blood aliquoted and stored at -80°C until IP1 analysis. Circulating levels of 2HG were measured at Agios Pharmaceuticals (Cambridge MA) using lLC-MS/MS analysis (AB Sciex 4000 Framingham MA) operating in bad electrospray mode. MRM data was acquired for each compound using the following transitions: 2HG (146.9/128.8 amu) BMS-794833 13 (151.9/133.8 amu) & 3HMG (160.9/98.9 amu). Chromatographic separation was performed using an ion-exchanged column (Bio-rad Fast Acid analysis 9 μm 7.8 mm × 100 mm; Bio-rad). The circulation rate BMS-794833 was 1 ml/min of 0.1% formic acid in water with a total run time of 4 minutes. 30 μl of sample was extracted by adding 30 μl of internal standard (ISTD) in water followed by 200 μl of acetonitrile. The sample was vortex combined centrifuged and 100 μL of supernatant transferred to a clean 96-well plate. The supernatant was diluted with 100 μl of deionized water and 25 μl injected on column. Statistical analysis The assessment of biomarkers with respect to mutational status or study site was performed using precise Mann-Whitney-Wilcoxon test. Median and interquartile ranges are provided as descriptive statistics. Correlations were quantified as Spearman’s correlation coefficients and tested with the Spearman’s test. P-values of <0.05 were considered statistically significant. Results Patient Characteristics The patient characteristics of the Screening and Validation cohorts are summarized in Table 1. A total of 31 diagnosed ICC individuals with clinically-determined and gene mutational status and with available banked whole blood comprised the Screening cohort. The median age of this group was 57 years and approximately 65% of these individuals presented with stage IV disease. These characteristics are consistent with individuals that normally undergo medical mutational profiling in an effort to identify alternate treatment programs after failing standard of care. Table 1 Characteristics of individuals with intrahepatic cholangiocarcinoma across cohorts. In order to increase analysis of ICC individuals a second Validation cohort consisting of 38 ICC individuals who underwent medical resection was then recognized and retrospectively genotyped to identify mutations. The age sex and CA19-9 blood levels of this Validation cohort were comparable to those in the Screening cohort (Table 1). However BMS-794833 the Validation group individuals spanned early disease phases including stage I (50%) stage II (~13%) and stage BMS-794833 III (37%) and did not include stage IV individuals. IDH1 and IDH2 mutational status In the Screening cohort mutations were found in 11 out of 31 individuals with ICC for an overall incidence of 35%. These included point mutations in p.R132C (n=7) p.R132L (n=3) and p.R132G (n=1) (Table 2 and Supplemental Table 1). No mutations were recognized. The ICC resected.

As a driver for many biological processes phosphorylation remains an area

As a driver for many biological processes phosphorylation remains an area of intense research interest. of experimental approaches. These methods included the use of synchronous precursor selection (SPS) to enhance TMT reporter ion Fostamatinib disodium intensity and accuracy. We found that (i) ratio distortion remained a problem for phosphopeptide analysis in multiplexed quantitative workflows (ii) ratio distortion can be overcome by the use of an SPS-MS3 scan (iii) interfering ions generally possessed a different charge state than the target precursor and (iv) selecting only the phosphate neutral loss peak (single notch) for the MS3 scan still provided accurate ratio measurements. Remarkably these data suggest that the underlying cause of interference may not be due to coeluting and cofragmented peptides but instead from consistent low level background fragmentation. Finally as a proof-of-concept 10-plex experiment we compared phosphopeptide levels from five murine brains to five livers. In total the SPS-MS3 method quantified 38?247 phosphopeptides corresponding to 11?000 phosphorylation sites. With 10 measurements recorded for each phosphopeptide this equates to Fostamatinib disodium more than 628?000 binary comparisons collected in less than 48 h. As a key mediator of cellular signaling phosphorylation remains a principal target for biological interrogation.1 Identifying and quantifying the phosphorylation state of proteins involved in cell progression metabolism growth and disease is critical for the continued elucidation of cellular function.2 Global phosphoproteome characterization is challenging due to the estimated large volume of phosphorylation sites in eukaryotic cells and the often low abundance/stoichiometry of the phosphoproteome.3 4 Continuing technological and methodological advancements have resulted in the characterization of tens of thousands of phosphorylation sites across numerous species but it is apparent that only a fraction of all phosphorylation events have been characterized.5?11 Furthermore phosphorylation dynamics assessed via relative quantification have historically been limited to binary or ternary comparisons further limiting the breadth and depth of phosphopeptide analysis.12?17 Novel methodologies are needed in order to overcome the current shortcomings of phosphoproteome characterization. Mass spectrometry remains an unmatched platform for comprehensive phosphoproteome analysis. Coupling deep identification with relative quantification has provided valuable biological insights that would be otherwise unobtainable by traditional biochemical techniques.18?24 Isobaric tags for relative and absolute quantitation (iTRAQ) and tandem-mass-tag (TMT) based methodologies permit the simultaneous comparison of up to 8 Fostamatinib disodium (iTRAQ) or 10 (TMT) samples facilitating complex experimental designs and the inclusion of biological replicates within the same experiment. A primary hurdle for isobaric based quantification technologies is the presence of interfering coisolated species that result in distorted reporter ion intensities. A number of publications have documented this phenomenon and several have demonstrated approaches to alleviate the interference.25?31 One such approach was the inclusion of a quantitative MS3 spectrum.32 Recently the sensitivity of the MS3 method was dramatically improved by isolating multiple fragment ions in the MS2 spectrum using isolation waveforms with multiple notches (e.g. synchronous precursor selection SPS).33 The SPS-MS3 method is available on the Orbitrap Fusion Fostamatinib disodium which leverages advancements in software and hardware to provide increased scan rates and improved sensitivity resolution and quantitative accuracy. Furthermore a unique architecture expands the concept of a hybrid mass spectrometer by incorporating three mass analyzers (i.e. quadrupole mass filter quadrupole ion trap and Orbitrap) operating in a task parallelized manner. Here we IP1 assessed the performance of the SPS-MS3 method on two different phosphoproteome samples. We utilized a 2-phosphoproteome model of interference to characterize the quantitative accuracy of various SPS-MS3 and MS2 methods Fostamatinib disodium on the Orbitrap Fusion. We observed that known ratios were distorted for the MS2 method compared to the SPS-MS3 method. In a large-scale demonstration of the method we performed a proteome-wide.