Mass Spectrometry (MS) is a common method for identification of proteins.
MS is particularly helpful for protein-protein interaction assays or interactome studies, which aim to identify the interacting partners of a protein within a cell at a time point. Here, in so called affinity purification MS (AP/MS) or capture MS experiments, the protein of interest and its interaction partners are isolated by co-immunoprecipitation (Co-IP).
The subsequent identification of the interacting partners by MS is called bottom-up proteomics. The protein samples are specially prepared for MS analysis: the samples are denatured, reduced, alkylated, and digested. Then, the resulting peptides are cleaned-up and transferred to the MS instrument
Nano-Traps for IP-MS
Nano-Traps are beneficial for MS analysis following Co-IP, because of their reliable performance, high affinity, and low background of protein contaminations. The superior stability of the Nano-Traps allows to apply stringent washing conditions. In most cases, even buffers containing chaotropic reagents can be used, whereas these would inactivate other IgG-based or Streptavidin-based affinity resins. Therefore, background can be further reduced, which increases the sensitivity of the sample analysis. This may be required for analysis of some post-translational modifications (PTMs).
It is recommended to conduct the MS sample preparation on-bead to avoid loss of sample by incomplete elution from the Nano-Trap.
Immunoprecipitation mass spectrometry (IP-MS) is also called affinity purification mass spectrometry (AP-MS) or affinity capture mass spectrometry (affinity capture MS).PRODUCTS
On-bead digestion with Nano-Traps
It is strongly recommended to conduct the sample preparation for MS analysis on-bead, which is a standard application. Using “on-bead digestion”, more interaction partners are transferred to MS analysis, whereas the elution from bead may cause loss of sample, e.g. by incomplete elution.
Because of their small size, the digestion of a single domain antibody bound to a Nano-Trap yields in only 4-7 peptides. For example, the GFP-Nanobody of GFP-Trap yields in only 4-5 peptides after trypsination. This small number of peptides does not add complexity to the MS analysis of the total protein sample.