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Related Concept Videos

Next-generation Sequencing03:00

Next-generation Sequencing

The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
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RNA-seq03:21

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Maxam-Gilbert Sequencing01:05

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Reduction of Alkynes to trans-Alkenes: Sodium in Liquid Ammonia02:10

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Related Experiment Video

Updated: Jun 22, 2026

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications
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Application of a coupled reducing system to Edman sequencing.

F Rusnak1, G Hathaway

  • 1California Institute of Technology, The Beckman Institute, Pasadena, CA 91125, USA.

Journal of Biomolecular Techniques : JBT
|June 6, 2009
PubMed
Summary
This summary is machine-generated.

This study introduces a combined reducing system using tris-(2-carboxyethyl)-phosphine hydrochloride and dithiothreitol for improved protein sequencing. The new method enhances quantitation of phenylthiohydantoin-Asp and increases repetitive yield in beta-lactoglobulin sequencing.

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Protein Chemistry

Background:

  • Dithiothreitol (DTT) is a common reducing agent in protein sequencing.
  • Oxidation products of DTT can interfere with sensitive quantitation.
  • Improved reducing systems are needed for higher accuracy in protein analysis.

Purpose of the Study:

  • To develop a coupled reducing system to minimize interference from DTT oxidation products.
  • To enhance the quantitation of phenylthiohydantoin-Asp (PTH-Asp).
  • To improve the repetitive yield in protein sequencing of beta-lactoglobulin.

Main Methods:

  • Addition of tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP) to Applied Biosystems' R4A and R5 reagents.
  • Coupling TCEP with dithiothreitol (DTT) to create a combined reducing system.
  • Quantitation of phenylthiohydantoin-Asp (PTH-Asp) using the enhanced system.

Main Results:

  • The coupled TCEP-DTT system eliminated or significantly reduced interference from DTT oxidation products.
  • Quantitation of PTH-Asp was achieved below the 400-fmol level.
  • A measurable increase in repetitive yield for beta-lactoglobulin sequencing was observed.
  • Instrument PTH standards demonstrated stability for over 3 months.

Conclusions:

  • The coupled TCEP-DTT reducing system offers superior performance for protein sequencing.
  • This method enhances sensitivity and accuracy in amino acid quantitation.
  • The improved system contributes to more reliable and efficient protein analysis.