Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

Peptide Identification Using Tandem Mass Spectrometry01:33

Peptide Identification Using Tandem Mass Spectrometry

6.7K
Tandem mass spectrometry, also known as MS/MS or MS2, is an analytical technique that employs two mass analyzers. Essentially it is a series of mass spectrometers that helps isolate a particular biomolecule and then helps study its chemical properties.
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...
6.7K

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Deciphering cytokine-driven ADP-ribosylation signaling networks via Af1521-based mass spectrometry analysis of labile Glu/Asp-linkages.

Nature communications·2026
Same author

Benchmarking Plasma Proteomics Workflows and Their Correlation to Clinical Routine Protein Assays.

Journal of proteome research·2026
Same author

Glucocorticoid-Induced Proteome and Phosphoproteome Changes in Breast Cancer Cell Lines.

Journal of proteome research·2026
Same author

The Japanese Archipelago sheltered cave lions, not tigers, during the Late Pleistocene.

Proceedings of the National Academy of Sciences of the United States of America·2026
Same author

Comparative Analysis of Lysine-Specific Peptidases for Optimizing Proteomics Workflows.

Journal of proteome research·2025
Same author

New methods on the block: Taxonomic identification of archaeological bones in resin-embedded sediments through paleoproteomics.

PNAS nexus·2025

Related Experiment Video

Updated: Sep 7, 2025

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis
05:47

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Published on: June 25, 2020

5.3K

Optimal analytical strategies for sensitive and quantitative phosphoproteomics using TMT-based multiplexing.

Claire Koenig1, Ana Martinez-Val1, Giulia Franciosa1

  • 1Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark.

Proteomics
|June 17, 2022
PubMed
Summary

For deep phosphoproteome coverage in mass spectrometry, microflow fractionation (MF) is best for >5 μg peptide input, while stage-tip fractionation (STF) excels for <5 μg. This guides optimal workflow selection based on sample amount.

Keywords:
high-pH fractionationisobaric labelingphosphoproteomicsscale-downtandem mass tags

More Related Videos

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis
07:44

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis

Published on: June 8, 2020

12.8K
A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors
10:17

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Published on: April 29, 2022

2.5K

Related Experiment Videos

Last Updated: Sep 7, 2025

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis
05:47

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Published on: June 25, 2020

5.3K
TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis
07:44

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis

Published on: June 8, 2020

12.8K
A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors
10:17

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Published on: April 29, 2022

2.5K

Area of Science:

  • Proteomics
  • Quantitative Mass Spectrometry
  • Biochemistry

Background:

  • Large-scale phosphoproteomics relies on mass spectrometry (MS) for protein phosphorylation site identification.
  • Tandem mass tags (TMTs) and high-pH reversed-phase peptide fractionation enhance coverage in quantitative MS.
  • Limited sample amounts can impact sensitivity and dynamic range in phosphoproteomic analyses.

Purpose of the Study:

  • To benchmark TMT-based fractionation strategies against label-free quantification with data-independent acquisition (LFQ-DIA).
  • To investigate the impact of varying peptide input amounts on TMT-fractionation efficiency in phosphoproteomics.
  • To compare microflow fractionation (MF) and stage-tip fractionation (STF) for phosphopeptide enrichment.

Main Methods:

  • Quantitative mass spectrometry (MS)-based phosphoproteomics.
  • Tandem mass tags (TMTs) labeling and offline high-pH reversed-phase peptide chromatographic fractionation.
  • Comparison of microflow (MF) and stage-tip fractionation (STF) strategies.
  • Benchmarking against single-shot label-free quantification with spectral library-free data independent acquisition (LFQ-DIA).
  • Systematic scaling of peptide input from 12.5 to 1 μg per sample.

Main Results:

  • Microflow fractionation (MF) with TMT labeling provides the deepest phosphoproteome coverage for input amounts >5 μg/sample.
  • Stage-tip fractionation (STF) of enriched phosphopeptides is optimal for lower input amounts (<5 μg/peptide/sample).
  • TMT-based MF achieved greater depth than single-shot LFQ-DIA across tested conditions.

Conclusions:

  • Workflow choice in phosphoproteomics depends critically on available sample amount.
  • MF is recommended for higher sample inputs (>5 μg) for maximal phosphoproteome coverage.
  • STF is the preferred method for limited sample amounts (<5 μg), optimizing sensitivity and coverage.