Peptide Isolation

Isolation and Detection of low concentrated Peptides using
patented MAB-SensBeads® and Mass Spectrometry

Advances for the isolation of low concentrated peptides when using MAB-SensBeads® combined with mass spectrometry:

  • high sensitivity
  • short isolation time
  • no streptavidin-coated beads and biotinylated antibodies necessary
  • no protein A/G-beads and crosslinking necessary

Isolation of Insulin from serum using different magnetic particles coupled with Insulin antibody and detection by LC-MS.

Tested magnetic particles and methods:

  1. MAB-SensBeads® (activated) + Insulin antibody
  2. MAB-SensBeads® (activated) + anti-Fcγ antibody + Insulin antibody + crosslinking
  3. MAB-SensBeads® (activated) + Protein G + Insulin antibody + crosslinking
  4. Commercial hydrophilic particles (activated) + Insulin antibody
  5. Commercial hydrophilic particles + Streptavidin + monobiotinylated
    Insulin antibody
  6. Commercial hydrophilic particles + Protein G + Insulin antibody + crosslinking
  7. Commercial latex particles + Streptavidin + monobiotinylated Insulin antibody
  8. Commercial latex particles + Protein G +  Insulin antibody + crosslinking
  9. Commercial agarose beads (activated) + anti-Fcγ antibody +
    Insulin antibody + crosslinking

DATA:

 

Using activated MAB-SensBeads® for immobilization of anti-Insulin antibody results in the highest readout for insulin during mass spectrometric analysis.

The next highest readouts can be achieved using either latex particles coupled with Protein G (Distributor 3) and anti-Insulin antibody or latex particles coupled with streptavidin (Distributor 2) and monobiotinylated anti-Insulin antibody (see Nr. 8).

Additional investigations showed, when performing immunopurification of Prostate Specific Antigen, that anti-PSA antibody is coeluting from the streptavidin- or Portein A/G-coated particles during the elution step (see Immunopurification).
This antibody bleeding results in a severe loss of antibody and results in false readouts.

The novel texture of the MAB-SensBeads® and direct antibody coupling allows the same detection sensitivity, as compared to other particles, without using streptavidin coatings and biotinylated antibodies or Protein A/G coatings followed by subsequent crosslinking after antibody immobilization.

With the MAB-SensBeads®, this drawbacks can be obviated.