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: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:* To compare surgical and oncological outcomes in patients underwent to colorectal resection with 3D vs 2D laparoscopic technique.
* To evaluate the visual overload in surgeons using 3D laparoscopic technique.
Project description:Global shotgun proteomic analysis using data independent acquisition (DIA) and Spectronaut analysis on the input cell lysates before the enrichment step in proximity labeling experiments, specifically MCF10A cells stably expressing miniTurbo-BAD in 3D with biotin treatment (n=4)
Project description:We developed a method to estimate the 3D interaction probabilities of chromatin loops across the genome on an absolute scale from Micro-C maps. To calibrate the method, we performed Micro-C on two engineered mouse embryonic stem cell (mESC) lines, each containing a fluorescently labeled chromatin loop that was quantified in previous live imaging studies. One loop is an endogenous loop containing the Fbn2 gene, and the other is a synthetic loop near the Npr3 gene. We performed two replicates of Micro-C per cell line. Using our absolute quantification method, we find that loops generally form with low probabilities. We also provide an ultra-deep merged Micro-C map for mESCs that combines all existing mESC Micro-C datasets to date, containing a total of 15.6 billion unique interactions.
Project description:Collagen is the most abundant protein in mammals and a major structural component of the extracellular matrix (ECM). Changes to ECM composition occur as a result of numerous physiological and pathophysiological causes, and a common means to evaluate these changes is the collagen 3 (Col3) to collagen 1 (Col1) ratio. Current methods to measure the Col3/1 ratio suffer from a lack of specificity and often under- or over-estimate collagen composition and quantity. This manuscript presents a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of Col3 and Col1 in FFPE tissues. Using surrogate peptides to generate calibration curves, Col3 and Col1 are readily quantified in FFPE tissue sections with high accuracy and precision. The method is applied to several tissue types from both human and reindeer tissues, demonstrating its generalizability. In addition, the targeted LC-MS/MS method permits quantitation of the hydroxyprolinated form of Col3, which has significant implications for understanding not only the quantity of Col3 in tissue, but also understanding of the pathophysiology underlying many causes of ECM changes. This manuscript presents a straight forward, accurate, precise and generalizable method for quantifying the Col3/1 ratio in a variety of tissue types and organisms.