Project description:Mass spectrometry (MS)-based proteomics is known for its high accuracy in quantifying peptides and proteins using various calibration strategies, including internal and external calibration curves. While external multi-point calibration curves are created from serial dilutions, they often fail to account for sample-specific matrix effects. In contrast, internal calibration curves account for sample matrix but face scalability and cost challenges for whole proteome analyses. In this manuscript we present a novel TMT-based multipoint internal calibration curve strategy, referred to as TMTCal, which enables the generation of internal calibration curves for all peptides identified within a proteome within a single experiment. We applied this strategy to human ovarian cancer cells to evaluate the linear quantitative responses of all the identified peptides and reveal the significant proteome changes associated with cisplatin treatment.
Project description:In this study, we developed and optimized a nanoproteomic workflow that we termed Nanogram TMT Processing in One Tube (NanoTPOT). Through the assessment of proteolytic digestion, tandem mass tag (TMT) labeling, online and offline frac-tionation strategies, our optimized workflow effectively eliminated the need for sample desalting and enabled compatible sample processing for MS analysis. We further applied the NanoTPOT workflow to examine cellular response to stress caused by dithiothreitol in Hela cells, where we identified and quantified 6935 proteins in a TMT 10-plex experiment with one microgram of starting material in each channel.
Project description:By reporting molar abundances of proteins, absolute quantification determines their stoichiometry in complexes, pathways or networks and also relates them to abundances of non-protein biomolecules. Typically, absolute quantification relies either on protein- specific isotopically labelled peptide standards or on a semi-empirical calibration against the average abundance of peptides chosen from arbitrary selected standard proteins. Here we developed a generic protein standard FUGIS (Fully unlabelled Generic Internal Standard) that requires no isotopic labelling, synthesis of standards or external calibration and is applicable to proteins of any organismal origin. FUGIS is co-digested with analysed proteins and enables their absolute quantification in the same LC-MS/MS run. By using FUGIS, median based absolute quantification (MBAQ) workflow provides similar quantification accuracy compared to isotopically-labelled peptide standards and outperforms methods based on external calibration or selection of best ionized reporter peptides (Top3 quantification) with a median quantification error less than 15%
Project description:Calibration array used to assess probe specific binding behaviour across eight different amounts of DNA starting material. This calibration step was performed to select one best out of three probes per gene based on probe responsiveness on increasing DNA staring material. It was further used to calibrate a subsequent microarray experiment to account for probe specific binding behaviour as part of the normalization process (see also Dennenmoser et al. 2017 Copy number increases of transposable elements and protein coding genes in an invasive fish of hybrid origin).
Project description:Despite the widespread adoption of ChIP-seq there is still no consensus on quality assessment metrics. No single published metric can reliably discriminate the success or failure of an experiment, thus hampering objectivity and reproducibility of quality control. We introduce a new framework for ChIP-seq data quality assessment that overcomes the limitation of previous solutions. Our tool called "ChIC" incorporates a novel set of quality control metrics integrated into one single score summarizing the sample quality and a reference compendium with thousands of published ChIP-seq samples, for easier evaluation of new data. This test dataset contain an example of succesfull and non-succesfull ChIP-seq sample for mouse H3K27me3.
Project description:Assessment of technical error in a dual-channel, two timepoint experiment using White lab Drosophila melanogaster microarrays Keywords: repeat sample
Project description:We propose a fully automated novel workflow for lipidomics based on flow injection, followed by liquid chromatography-high-resolution mass spectrometry (FI/LC-HRMS). The workflow combined in-depth characterization of the lipidome achieved via reversed-phase LC-HRMS with absolute quantification by using a large number of lipid species-specific and/or retention time (RT)-matched/class-specific calibrants. The lipidome of 13C-labelled yeast (LILY) provided a large panel of cost-effective internal standards (ISTDs) covering triacylglycerols (TG), steryl esters (SE), free fatty acids (FA), diacylglycerols (DG), sterols (ST), ceramides (Cer), hexosyl ceramides (HexCer), phosphatidylglycerols (PG), phosphatidylethanolamines (PE), phosphatidic acids (PA), cardiolipins (CL), phosphatidylinositols (PI), phosphatidylserines (PS), phosphatidylcholines (PC), lysophosphatidylcholines (LPC) and lysophosphatidylethanolamines (LPE). The workflow in combination with the LILY lipid panel enables simultaneous quantification via (1) external multi-point calibration with internal standardization and (2) internal one-point calibration with LILY as a surrogate ISTD, increasing the coverage while keeping the accuracy and throughput high. Extensive measures on quality control allowed us to rank the calibration strategies and to automatically select the calibration strategy of the highest metrological order for the respective lipid species. Overall, the workflow enabled a streamlined analysis, with a limit of detection in the low femtomolar range, and provided validation tools together with absolute concentration values for >350 lipids in human plasma on a species level. Based on the selected standard panel, lipids from 7 classes (LPC, LPE, PC, PE, PI, DG, TG) passed stringent quality filters, which included QC accuracy, a precision and recovery bias of <30% and concentrations within the 99% confidence interval of the international laboratory comparison of SRM 1950, NIST, USA. The quantitative values are independent of common deuterated or non-endogenous ISTDs, thus offering cross-validation of different lipid methods and further standardizing lipidomics.