MS<sup>3</sup>‐based quantitative proteomics using pulsed‐Q dissociation

Cao, Zhiyun; Robinson, Renã A. S.

doi:10.1002/rcm.7192

Cited by 4 publications

(4 citation statements)

References 32 publications

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“…However, suppression of iTRAQ protein ratios in MS/MS experiments is a noted limitation of this quantitative approach . Proteomics workflows to handle suppression issues at the MS 3 level have been recently reported. − …”

Section: Resultsmentioning

confidence: 99%

Proteomics Reveals Age-Related Differences in the Host Immune Response to Sepsis

et al. 2013

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Sepsis is commonly caused by community-acquired pneumonia (CAP) and may develop into severe sepsis, characterized by multiple organ failure. The risk of severe sepsis among CAP patients and subsequent mortality increases sharply after the age of 65. The molecular mechanisms associated with this age-related risk are not fully understood. To better understand factors involved with increased incidence and mortality of severe sepsis in the elderly, we used a nested case-control study of patients enrolled in a multicenter observational cohort of 2,320 participants with CAP. We identified a total of 39 CAP patients 50-65 and 70-85 years old who did or did not develop severe sepsis. Plasma samples were obtained on presentation to the emergency department and prior to therapeutic interventions. A semi-quantitative plasma proteomics workflow was applied which incorporated tandem immunoaffinity depletion, iTRAQ labeling, strong cation exchange fractionation, and nanoflow-liquid chromatography coupled to high resolution mass spectrometry. In total, 772 proteins were identified, of which, 58 proteins exhibit statistically significant differences in expression levels amongst patients with severe sepsis as a function of age. Differentially-expressed proteins are involved in pathways such as acute phase response, coagulation signaling, atherosclerosis signaling, lipid metabolism, and production of nitric oxide and reactive oxygen species. This study provides insight into factors that may explain age-related differences in incidence of severe sepsis in the elderly.

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Section: Resultsmentioning

confidence: 99%

Proteomics Reveals Age-Related Differences in the Host Immune Response to Sepsis

et al. 2013

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“…This method, similarly to other multiplexing methods, allows for the analysis of multiple samples at once, and is applicable to samples originating from cells, tissues, bodily fluids, or whole organisms. In addition, cPILOT labeled peptides are able to be analyzed using either a low 24 or high resolution (60,000) instrument. In comparison, obtaining successful measurement using other multiplexing methods may be limited to a specific type of sample ( i.e.…”

Section: Discussionmentioning

confidence: 99%

“…MS 3 acquisition with a top-ion 11 or selective-y 1 -ion method 15 can help reduce reporter ion interference and improve quantitative accuracy of cPILOT. The use of MS 3 in the acquisition method requires a high-resolution instrument with an orbitrap mass analyzer although low resolution ion trap instruments may also work 24 .…”

Section: Introductionmentioning

confidence: 99%

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

King

Dudenhoeffer

Gu³

et al. 2017

JoVE

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There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies that allow simultaneous measurement of multiple samples have become widespread and greatly reduce experimental costs and times. Our laboratory developed a technique called combined precursor isotopic labeling and isobaric tagging (cPILOT), which enhances sample multiplexing of traditional isotopic labeling or isobaric tagging approaches. Global cPILOT can be applied to samples originating from cells, tissues, bodily fluids, or whole organisms and gives information on relative protein abundances across different sample conditions. cPILOT works by 1) using low pH buffer conditions to selectively dimethylate peptide N-termini and 2) using high pH buffer conditions to label primary amines of lysine residues with commercially-available isobaric reagents (see Table of Materials/Reagents). The degree of sample multiplexing available is dependent on the number of precursor labels used and the isobaric tagging reagent. Here, we present a 12-plex analysis using light and heavy dimethylation combined with six-plex isobaric reagents to analyze 12 samples from mouse tissues in a single analysis. Enhanced multiplexing is helpful for reducing experimental time and cost and more importantly, allowing comparison across many sample conditions (biological replicates, disease stage, drug treatments, genotypes, or longitudinal time-points) with less experimental bias and error. In this work, the global cPILOT approach is used to analyze brain, heart, and liver tissues across biological replicates from an Alzheimer's disease mouse model and wild-type controls. Global cPILOT can be applied to study other biological processes and adapted to increase sample multiplexing to greater than 20 samples.

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“…cPILOT doubles the multiplexing capacity of isobaric tags alone and was most recently demonstrated at 24-plex with DiLeu tags (106). cPILOT has been applied to several models of aging and disease (5,104,(106)(107)(108)(109), oxidative posttranslational modifications (102,108,110), and low-resolution mass spectrometers (111). Recently, we increased the experimental throughput of cPILOT to 32-plex with robotic liquid handlers to study an early-stage diabetes model (112,113).…”

Section: Enhanced Multiplexing Strategiesmentioning

confidence: 99%

Enhanced Multiplexing Technology for Proteomics

Bowser

Robinson

2023

Annual Rev. Anal. Chem.

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The identification of thousands of proteins and their relative levels of expression has furthered understanding of biological processes and disease and stimulated new systems biology hypotheses. Quantitative proteomics workflows that rely on analytical assays such as mass spectrometry have facilitated high-throughput measurements of proteins partially due to multiplexing. Multiplexing allows proteome differences across multiple samples to be measured simultaneously, resulting in more accurate quantitation, increased statistical robustness, reduced analysis times, and lower experimental costs. The number of samples that can be multiplexed has evolved from as few as two to more than 50, with studies involving more than 10 samples being denoted as enhanced multiplexing or hyperplexing. In this review, we give an update on emerging multiplexing proteomics techniques and highlight advantages and limitations for enhanced multiplexing strategies. Expected final online publication date for the Annual Review of Analytical Chemistry, Volume 16 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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MS³‐based quantitative proteomics using pulsed‐Q dissociation

Abstract: PQD-MS(3) methods increase the amount of MS/MS spectra collected and number of quantified proteins and are accessible to those researchers with not only an orbitrap but also an ion trap mass spectrometer.

Cited by 4 publications

References 32 publications

Proteomics Reveals Age-Related Differences in the Host Immune Response to Sepsis

Proteomics Reveals Age-Related Differences in the Host Immune Response to Sepsis

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

Enhanced Multiplexing Technology for Proteomics

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MS3‐based quantitative proteomics using pulsed‐Q dissociation

Abstract: PQD-MS(3) methods increase the amount of MS/MS spectra collected and number of quantified proteins and are accessible to those researchers with not only an orbitrap but also an ion trap mass spectrometer.

Cited by 4 publications

References 32 publications

Proteomics Reveals Age-Related Differences in the Host Immune Response to Sepsis

Proteomics Reveals Age-Related Differences in the Host Immune Response to Sepsis

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

Enhanced Multiplexing Technology for Proteomics

Contact Info

Product

Resources

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MS³‐based quantitative proteomics using pulsed‐Q dissociation