Optimizing the surface plasmon resonance/mass spectrometry interface for functional proteomics applications : How to avoid and utilize nonspecific adsorption

• A great challenge in functional or interaction proteomics is to map protein networks and establish a functional relationship between expressed proteins and their effects on cellular processes. These cellular processes can be studied by characterizing binding partners to a "bait" protein against a complex background of other molecules present in cells, tissues, or biological fluids. This so-called ligand fishing process can be performed by combining surface plasmon resonance biosensors with MS. This combination generates a unique and automated method to quantify and characterize biomolecular interactions, and identify the interaction partners. A general problem in chip-based affinity separation systems is the large surface-to-volume ratio of the fluidic system. Extreme care, therefore, is required to avoid nonspecific adsorption, resulting in losses of the target protein and carry-over during the affinity purification process, which may lead to unwanted signals in the final MS analysis and a reduction in sensitivity. In this study, carry-over of protein and low-molecular weight substances has been investigated systematically and cleaning strategies are presented. Furthermore, it is demonstrated that by the introduction of colloidal particles as a capturing and transporting agent, the recovery yield of the affinity-purified ligand could be improved nearly twofold.

Subject headings

NATURVETENSKAP  -- Biologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences (hsv//eng)

Keyword

Interactions

Mass spectrometry

Proteins

Surface plasmon resonance

adsorption

article

functional proteomics

instrumentation

matrix assisted laser desorption ionization time of flight mass spectrometry

priority journal

separation technique

technique

Animals

Biosensing Techniques

Cattle

Ligands

Protein Array Analysis

Proteomics

Reproducibility of Results

Serum Albumin

Publication and Content Type

ref (subject category)

art (subject category)