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Related Concept Videos

Peptide Identification Using Tandem Mass Spectrometry01:33

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...
Tandem Mass Spectrometry01:21

Tandem Mass Spectrometry

Tandem mass spectrometry is a technique that uses multiple mass analyzers in series to obtain a higher selectivity and reduce chemical noise during analyte detection. Instruments with multiple analyzers separated by an interaction cell enable secondary fragmentation and selected study of the fragment ions.Secondary fragmentations occur in the interaction cell and can be induced by various factors. Fragmentation induced by collision with inert gases, such as N2, Ar, He, etc., is called...
Ion-Exchange Chromatography01:09

Ion-Exchange Chromatography

Ion-exchange chromatography, or IEC, is a technique for separating ions based on their affinity for the stationary phase. The stationary phase is a cross-linked polymer resin with covalently attached ionic functional groups. The functional groups can be either positively charged (cation exchangers) or negatively charged (anion exchangers). A cation exchanger consists of a polymeric anion and active cations, while an anion exchanger is a polymeric cation with active anions. The choice of...

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Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
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Enhancing Proteome Coverage by Using Strong Anion-Exchange in Tandem with Basic-pH Reversed-Phase Chromatography for

Tian Zhang1, Xinyue Liu1, Valentina Rossio1

  • 1Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, United States.

Journal of Proteome Research
|November 14, 2023
PubMed
Summary

This study introduces a new peptide fractionation method using strong anion-exchange (SAX) chromatography before basic pH reversed-phase (BPRP) separation. This enhanced proteomic analysis strategy increases protein and unique peptide identification in complex samples.

Keywords:
FAIMSRTSSAXSPS-MS3TMTproeclipse

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Proteomic studies require effective fractionation to maximize proteome coverage.
  • Tandem Mass Tag (TMT) experiments, while powerful, necessitate fractionation for comprehensive protein identification.

Purpose of the Study:

  • To develop and validate a simple, two-step peptide fractionation strategy combining strong anion-exchange (SAX) and basic pH reversed-phase (BPRP) chromatography.
  • To improve proteome coverage and identify more unique peptides in TMT-based proteomic analyses.

Main Methods:

  • A novel two-step peptide fractionation protocol using SAX spin columns followed by BPRP fractionation.
  • Application of the strategy to a TMTpro18-plex experiment with nine human cell lines.
  • Comparison of protein and peptide quantification between SAX-partitioned and BPRP-only fractionation datasets.

Main Results:

  • The combined SAX-partition and BPRP fractionation strategy identified 10% more proteins and 20% more unique peptides compared to BPRP alone.
  • Noticeable differences in peptide charge and isoelectric point distribution were observed between SAX partitions.
  • Similar overall protein and peptide quantification numbers were achieved across different fractionation approaches.

Conclusions:

  • The proposed SAX-partition followed by BPRP fractionation is an effective method to enhance proteome coverage in TMT-based proteomics.
  • This strategy offers a valuable improvement for deep proteome profiling and biomarker discovery.
  • An online resource of protein abundance profiles across nine human cell lines is provided.