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Related Concept Videos

Capillary Electrophoresis: Applications01:30

Capillary Electrophoresis: Applications

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Capillary electrophoretic separations offer various modes, each with unique applications. These modes include capillary zone electrophoresis, capillary gel electrophoresis, capillary array electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, micellar electrokinetic chromatography, and capillary electrochromatography.
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Ion-Exchange Chromatography01:09

Ion-Exchange Chromatography

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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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Size-Exclusion Chromatography

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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.
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Higher molecular weight biomolecules are nonvolatile compounds that may decompose before ionizing or vaporizing during mass analysis with conventional electron impact ionization methods. Accordingly, electrospray ionization (ESI) is the favored method for vaporizing and ionizing biomolecules as it circumvents rapid fragmentation and enables the recording of mass signals for the entire biomolecule.
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The mass analyzer is a crucial component of the mass spectrometer. In the ionization chamber, the vaporized sample is bombarded with a high-energy electron beam to generate a radical cation and further fragment into neutral molecules, radicals, and cations. A series of negatively charged accelerator plates accelerate the cations into the mass analyzer. The mass analyzer separates ions according to their mass-to-charge (m/z) ratios and then directs them to the detector. The common types of mass...
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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.
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Expanding Differential Ion Mobility Separations into the MegaDalton Range.

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High-definition field asymmetric ion mobility spectrometry (FAIMS) coupled with advanced mass spectrometry now analyzes megadalton biomolecular assemblies. This breakthrough enables the study of large protein complexes and viruses previously beyond analytical reach.

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Area of Science:

  • Analytical Chemistry
  • Biochemistry
  • Mass Spectrometry

Background:

  • Ion mobility separations (IMS) and mass spectrometry (MS) have advanced for analyzing larger biomolecules.
  • Electrospray ionization (ESI) and native MS enabled analysis of proteins up to 100 kDa and complexes/viruses up to 10 MDa.
  • Differential FAIMS offers orthogonal separation but was limited to ions under 300 kDa.

Purpose of the Study:

  • To integrate high-definition FAIMS with a high-capacity Orbitrap mass spectrometer (Q-Exactive Orbitrap UHMR).
  • To extend the mass range of FAIMS analysis for very large biomolecular assemblies.
  • To demonstrate the capability for analyzing megadalton (MDa) ions in the native ESI regime.

Main Methods:

  • Integration of high-definition FAIMS with the Q-Exactive Orbitrap UHMR mass spectrometer.
  • Utilized native ESI for ionization of large biomolecules.
  • Size-selected oligomers of monoclonal antibody adalimumab (148 kDa).

Main Results:

  • Successfully analyzed nonamers (1.34 MDa) of adalimumab with mass-to-charge (m/z) up to ~17,000.
  • Demonstrated the survival and efficient separation of noncovalent MDa assemblies within the FAIMS process.
  • The UHMR mass spectrometer handled ions with m/z up to 80,000 and MDa-size ions.

Conclusions:

  • High-definition FAIMS coupled with the Q-Exactive Orbitrap UHMR enables analysis of the heaviest macromolecules.
  • This technological advancement opens new avenues for studying large, noncovalent biomolecular assemblies.
  • Expands the scope of IMS/MS for characterizing complex biological systems.