Related Experiment Video
Updated: Aug 8, 2026
06:09
Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry
Published on: July 31, 2011
Boundary between protein and peptide shown by their chromatographic behavior
Noriyuki Nimura1, Hiroko Itoh, Hiroshi Homma
1School of Pharmaceutical Sciences, Kitasato University, 9-1 Shirokane-5, Minato-ku, Tokyo 108-8641, Japan. nimura@chiral.pharm.kitasato-u.ac.jp
Summary
This study clarifies reversed-phase HPLC separation mechanisms for peptides and proteins. A new "Transitional Desorption Mode" explains how separation changes with molecular weight, bridging partition and elution modes.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Chromatography
Background:
- Reversed-phase High-Performance Liquid Chromatography (RP-HPLC) is crucial for separating peptides and proteins.
- Existing models do not fully explain RP-HPLC separation mechanisms across a wide range of polypeptide molecular weights.
Purpose of the Study:
- To systematically investigate and precisely explain the separation mechanisms of peptides and proteins in RP-HPLC.
- To identify the molecular weight transition point between different separation modes.
Main Methods:
- Utilized a nonporous octadecylsilyl (ODS) silica-gel column for systematic separation studies.
- Investigated a diverse range of peptides and proteins with varying molecular weights.
Main Results:
- Identified a critical molecular weight range (3500-4500) where separation mechanisms transition.
- Demonstrated that small peptides (<3000 MW) follow a partition mode, while larger proteins exhibit a characteristic elution mode.
Conclusions:
- Proposed a new separation mechanism, the "Transitional Desorption Mode", to explain RP-HPLC of polypeptides.
- This mode precisely accounts for the observed separation behavior across a broad spectrum of molecular weights.
More Related Videos
Related Concept Videos
01:13Principles Of Column Chromatography
The chromatography technique was first invented in 1901 by Michael S. Tswett, a Russian botanist, to separate plant pigments using organic solvents. Further, in 1941, Archer John Porter Martin and R. L. M. Synge modified the technique by packing silica gel into a column. A mixture of amino acids was then separated on the packed column using chloroform and water mixture as the mobile phase. This was the first report on column chromatography. At present, column chromatography is a widely used...
01:27SDS-PAGE
Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field.
A variation of gel electrophoresis, termed polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins...
A variation of gel electrophoresis, termed polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins...
01:22Two-dimensional Gel Electrophoresis
Two-dimensional gel electrophoresis is a high-resolution protein separation method first introduced by O' Farrell and Klose in 1975. This method involves protein separation by two dimensions, mass and charge, making it more accurate than one-dimensional gel electrophoresis.
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
Biological samples, such as cells...
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
Biological samples, such as cells...
01:18Chromatographic Methods: Terminology
Chromatography is an analytical technique widely used in fields such as chemistry, biology, environmental science, and pharmaceuticals to separate the components of a mixture and identify substances between them. The process of chromatography is based on the interactions between two distinct phases: the stationary phase and the mobile phase. The stationary phase is fixed in place by a supporting material, while the mobile phase moves over it, carrying the solutes. As the mobile phase travels,...
01:09Ion-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...
01:08Size-Exclusion Chromatography
In size-exclusion chromatography (SEC), also known as molecular-exclusion or gel-permeation chromatography, molecules are separated based on their sizes. This technique is important for separating large molecules such as polymers and biomolecules. The two classes of micron-sized stationary phases encountered in SEC are silica particles and cross-linked polymer resin beads. Both materials are porous, but their pore sizes vary significantly.
Silica particles offer advantages such as rigidity,...
Silica particles offer advantages such as rigidity,...