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:The comparison of GPSMs that's identified by StrucGP in 24 MouseBrain raw files, using MAP-Match and the conventional calibration method saparately.
Project description:Targeted LC/MS Metabolomics. Cecal contents were lyophilized overnight and approximately 2.5-5 mg was homogenized in 250 µL of 50% acidified acetonitrile (0.3% formic acid) for targeted LC/MS/MS metabolomics (acylcarnitines, amino acids, organic acids, nucleotides, and CoAs) according to validated, optimized protocols in our previously published study (Previs et al.). Separate aliquots of homogenates were extracted with solvents for each class of metabolites, and then each class was analyzed with a unique LC/MS/MS method to optimize their chromatographic resolution and sensitivity. Quantitation of metabolites in each assay module was achieved using multiple reaction monitoring of calibration solutions and study samples with isotopically-labelled internal standards on an Agilent 1290 Infinity UHPLC/6495 triple quadrupole mass spectrometer. Raw data was processed using Mass Hunter quantitative analysis software (Agilent). Calibration curves (R2 = 0.99 or greater) are either fitted with a linear or a quadratic curve with a 1/X or 1/X2 weighting. Metabolic and Proteomic Defects in Human Hypertrophic Cardiomyopathy Michael J. Previs, Thomas S. O’Leary, Neil B. Wood, Michael P. Morley, Brad Palmer, Martin LeWinter, Jaime Yob, Francis D. Pagani, Christopher Petucci, Min-Soo Kim, Kenneth B. Margulies, Zoltan Arany, Daniel P. Kelly, Sharlene M. Day bioRxiv 2021.08.18.455967; doi: https://doi.org/10.1101/2021.08.18.455967