Dataset Information

Discovery Proteomics and Absolute Protein Quantification Can Be Performed Simultaneously on an Orbitrap-Based Mass Spectrometer.

ABSTRACT: Mass spectrometry (MS) has steadily moved into the forefront of quantification-centered protein research. Protein cleavage isotope dilution MS is a proven way for quantifying proteins by using an isotope-labeled analogue of a peptide fragment of the parent protein as an internal standard. Parallel reaction monitoring (PRM) has become the go-to approach for such quantification on an Orbitrap-based instrument as it is assumed that the instrument sensitivity is enhanced. We performed a comparative study on data-dependent acquisition (DDA) and PRM-based workflows to quantify egg yolk protein precursors or vitellogenins (VTGs) Aa, Ab, and C in striped bass (Morone saxatilis). VTG proportions serve as a developmental measure of egg quality, possibly changing with the environment, and have been studied as an indicator of the health of North Carolina stocks. Based on single-factor analysis of variance comparisons of mean VTG amounts across fish from the same sample groupings, our results indicate that there is no statistical difference between MS1-based and MS2-based VTG quantification. We further conclude that DDA is able to deliver both discovery data and absolute quantification data in the same experiment.

SUBMITTER: Williams TI

PROVIDER: S-EPMC10077438 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Discovery Proteomics and Absolute Protein Quantification Can Be Performed Simultaneously on an Orbitrap-Based Mass Spectrometer.

Williams Taufika Islam TI Kowalchyk Cara C Collins Leonard B LB Reading Benjamin J BJ

ACS omega 20230322 13

Mass spectrometry (MS) has steadily moved into the forefront of quantification-centered protein research. Protein cleavage isotope dilution MS is a proven way for quantifying proteins by using an isotope-labeled analogue of a peptide fragment of the parent protein as an internal standard. Parallel reaction monitoring (PRM) has become the <i>go-to</i> approach for such quantification on an Orbitrap-based instrument as it is assumed that the instrument sensitivity is enhanced. We performed a compa ...[more]

PMID: 37033798

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Project description:There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. To date protein quantification in biological samples has been routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), and two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved so as to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring) and in MS/MS mode (parallel reaction monitoring). The ability of the quadrupole to select a restricted m/z range allows one to overcome the dynamic range limitations associated with trapping devices, and the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performance in terms of selectivity, dynamic range, and sensitivity. This high performance is further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60-min experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein quantification methods in complex samples and address the pressing demand of systems biology or biomarker evaluation studies.

Project description:Mass spectrometry (MS)-based proteomics is a powerful technology to globally profile protein abundances, activities, interactions, and modifications. The extreme complexity of proteomics samples, which often contain hundreds of thousands of analytes, necessitates continuous development of MS techniques and instrumentation to improve speed, sensitivity, precision, and accuracy, among other analytical characteristics. Here, we systematically evaluated the Orbitrap Ascend Tribrid mass spectrometer in the context of shotgun proteomics, and we compared its performance to that of the previous generation of Tribrid instruments─the Orbitrap Eclipse. The updated architecture of the Orbitrap Ascend includes a second ion-routing multipole (IRM) in front of the redesigned C-trap/Orbitrap and a new ion funnel that allows gentler ion introduction, among other changes. These modifications in Ascend hardware configuration enabled an increase in parallelizable ion injection time during higher-energy collisional dissociation (HCD) Orbitrap tandem MS (FTMS2) analysis of ∼5 ms. This enhancement was particularly valuable in the analyses of limited sample amounts, where improvements in sensitivity resulted in up to 140% increase in the number of identified tryptic peptides. Further, analysis of phosphorylated peptides enriched from the K562 human cell line yielded up to ∼50% increase in the number of unique phosphopeptides and localized phosphosites. Strikingly, we also observed a ∼2-fold boost in the number of detected N-glycopeptides, likely owing to the improvements in ion transmission and sensitivity. In addition, we performed the multiplexed quantitative proteomics analyses of TMT11-plex labeled HEK293T tryptic peptides and observed 9-14% increase in the number of quantified peptides. In conclusion, the Orbitrap Ascend consistently outperformed its predecessor the Orbitrap Eclipse in various bottom-up proteomic analyses, and we anticipate that it will generate reproducible and in-depth datasets for numerous proteomic applications.

Dataset Information

Discovery Proteomics and Absolute Protein Quantification Can Be Performed Simultaneously on an Orbitrap-Based Mass Spectrometer.

Publications

Discovery Proteomics and Absolute Protein Quantification Can Be Performed Simultaneously on an Orbitrap-Based Mass Spectrometer.

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