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Peptide Identification Using Tandem Mass Spectrometry

Peptide Identification Using Tandem Mass Spectrometry

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...

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Updated: Apr 10, 2026

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

Published on: May 4, 2022

An Augmented Multiple-Protease-Based Human Phosphopeptide Atlas.

Piero Giansanti¹, Thin Thin Aye¹, Henk van den Toorn¹

¹Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 Utrecht, the Netherlands; Netherlands Proteomics Centre, Padualaan 8, 3584 Utrecht, the Netherlands.

|June 16, 2015

Summary

This summary is machine-generated.

Mass spectrometry studies miss key regulatory sites due to trypsin digestion bias. Using diverse proteases reveals a broader human phosphoproteome, enhancing protein phosphorylation site detection by over 1,000-fold.

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A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

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

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

Published on: April 29, 2022

Area of Science:

Proteomics
Biochemistry
Molecular Biology

Background:

Mass spectrometry enables simultaneous monitoring of thousands of protein phosphorylation sites.
Current large-scale phosphoproteome studies often miss crucial regulatory phosphorylation sites.
This limitation is hypothesized to stem from the predominant use of trypsin digestion in sample preparation.

Purpose of the Study:

To investigate the impact of protease choice on phosphoproteome coverage.
To develop a less biased method for identifying protein phosphorylation sites.
To create a comprehensive human phosphopeptide atlas.

Main Methods:

Proteins were digested using multiple proteases, not solely trypsin.
Titanium dioxide (Ti(4+)) immobilized metal affinity capture (IMAC) was used for phosphopeptide enrichment.
Mass spectrometry (MS) was employed for phosphopeptide and phosphosite identification.

Main Results:

Employing multiple proteases significantly increased the detectable phosphoproteome compared to trypsin alone.
A comprehensive human phosphopeptide atlas was generated, containing 37,771 unique phosphopeptides and 18,430 unique phosphosites.
Fewer than one-third of identified phosphosites were detected across multiple protease datasets, highlighting protease-specific biases.
Each phosphorylation site was linked to a preferred protease, enhancing MS detectability.

Conclusions:

Tryptic bias is a significant limitation in current large-scale phosphoproteome studies.
Utilizing a panel of proteases substantially expands the coverage of the human phosphoproteome.
This protease-based strategy enhances the detection of specific protein phosphorylation sites by over 1,000-fold, improving the understanding of regulatory mechanisms.