Search research articles

ABOUT JoVE

Overview Leadership Blog JoVE Help Center

AUTHORS

Publishing Process Editorial Board Scope & Policies Peer Review FAQ Submit

LIBRARIANS

Testimonials Subscriptions Access Resources Library Advisory Board FAQ

RESEARCH

JoVE Journal Methods Collections JoVE Encyclopedia of Experiments Archive

EDUCATION

JoVE Core JoVE Business JoVE Science Education JoVE Lab Manual Faculty Resource Center Faculty Site

Terms & Conditions of Use

Related Concept Videos

You might also read

Related Articles

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

Sort by

Same author

Comparative genomic analysis of clinically relevant human skin-associated fungi.

Nature communications·2026

Same author

Proteomics reveals spatial and molecular heterogeneities in advanced atherosclerotic carotid artery plaques.

Nature cardiovascular research·2026

Same author

Author Correction: Regulatory T cells in the mouse hypothalamus control immune activation and ameliorate metabolic impairments in high-calorie environments.

Nature communications·2026

Same author

Cysteine availability tunes ubiquitin signaling via inverse stability of LRRC58 E3 ligase and its substrate CDO1.

Nature communications·2026

Same author

Deeper is not always better in plasma proteomics.

Nature biotechnology·2026

Same author

The 2025 Westlake Autumn Symposium for Al Proteomics and Virtual Cell.

Genomics, proteomics & bioinformatics·2026

Same journal

An assay to quantify sexual commitment and stage conversion in the human malaria parasite Plasmodium falciparum.

Nature protocols·2026

Same journal

Author Correction: Direct inoculation of bioreactor-controlled stirred suspension culture with cryopreserved human pluripotent stem cells.

Nature protocols·2026

Same journal

High-throughput measurements of protein domain functions using magnetic separation.

Nature protocols·2026

Same journal

Inducing physiological polarity and performing gene editing using CRISPR-Cas9 in human trophoblast organoids.

Nature protocols·2026

Same journal

Photocatalytic low-temperature defluorination of PTFE.

Nature protocols·2026

Same journal

Multimodal imaging and quantification of lanthanide chelate-labeled micro- and nanoplastics in plants.

Nature protocols·2026

See all related articles

Search research articles

Related Experiment Video

Updated: Jun 12, 2025

Proteomic Profile of EPS-Urine through FASP Digestion and Data-Independent Analysis

Published on: May 8, 2021

An accessible workflow for high-sensitivity proteomics using parallel accumulation-serial fragmentation (PASEF).

Patricia Skowronek¹, Georg Wallmann¹, Maria Wahle¹

¹Department Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.

Nature Protocols

|January 17, 2025

Summary

This summary is machine-generated.

This study presents a streamlined proteome analysis protocol using Evosep One chromatography and timsTOF mass spectrometry with parallel accumulation-serial fragmentation (PASEF) and data-independent acquisition (DIA) for routine, in-depth cellular analysis.

More Related Videos

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Published on: April 1, 2017

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Published on: August 6, 2018

Related Experiment Videos

Last Updated: Jun 12, 2025

Proteomic Profile of EPS-Urine through FASP Digestion and Data-Independent Analysis

Published on: May 8, 2021

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Published on: April 1, 2017

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Published on: August 6, 2018

Area of Science:

Proteomics
Mass Spectrometry
Biochemistry

Background:

Deep proteome analysis is vital for understanding cellular functions and diseases.
Current methods for routine proteome analysis lack depth and accuracy.
High-performance mass spectrometry is key to advancing proteomic insights.

Purpose of the Study:

To develop a robust and routine protocol for in-depth proteome analysis.
To combine chromatographic and mass spectrometric platforms for broad community use.
To optimize parameters for quantitative accuracy, specificity, and sensitivity in proteomic studies.

Main Methods:

Utilized Evosep One chromatography with pre-formed gradients and tip-based sample preparation.
Employed a trapped ion mobility time-of-flight mass spectrometer (timsTOF) with parallel accumulation-serial fragmentation (PASEF) and data-independent acquisition (DIA).
Used the py_diAID tool for optimizing PASEF and DIA method parameters, including isolation windows in mass-to-charge and ion mobility space.

Main Results:

Achieved reproducible quantification of 7,000 proteins in a human cancer cell line with 21-min injections.
Identified 29,000 phosphosites in phospho-enriched quadruplicates.
Demonstrated high quantitative reproducibility using Synchro-PASEF scan mode with Spectronaut or AlphaDIA analysis.

Conclusions:

The protocol enables routine, in-depth proteome coverage with high accuracy and sensitivity.
The combined Evosep One and timsTOF PASEF/DIA approach significantly enhances proteomic analysis efficiency.
This method offers a cost-effective and time-efficient solution for biological projects, requiring minimal hands-on time.