On-line bioaffinity-electrospray mass spectrometry for simultaneous detection, identification, and quantification of protein-ligand interactions.
We describe here an on-line combination of a surface acoustic wave (SAW) biosensor with electrospray ionization mass spectrometry (SAW-ESI-MS) that enables the direct detection, identification, and quantification of affinity-bound ligands from a protein-ligand complex on a biosensor chip. A trapping column was used between the SAW-biosensor and the electrospray mass spectrometer equipped with a micro-guard column, which provides simultaneous sample concentration and desalting for the mass spectrometric analysis of the dissociated ligand. First applications of the on-line SAW-ESI-MS combination include (1), differentiation of β-amyloid (Aβ) epitope peptides bound to anti-Aβ antibodies; (2), the identification of immobilized Substance P peptide-calmodulin complex; (3), identification and quantification of the interaction of 3-nitrotyrosine-modified peptides with nitrotyrosine-specific antibodies; and (4), identification of immobilized anti-α-synuclein-human α-synuclein complex. Quantitative determinations of protein-ligand complexes by SAW yielded dissociation constants (K(D)) from micro-to low nanomolar sample concentrations. The on-line bioaffinity-ESI-MS combination presented here is expected to enable broad bioanalytical application to the simultaneous, label-free determination and quantification of biopolymer-ligand interactions, as diverse as antigen-antibody and lectin-carbohydrate complexes.
Dragusanu
Mihaela
M
Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry, University of Konstanz, Konstanz, Germany.
Petre
Brîndusa-Alina
BA
Slamnoiu
Stefan
S
Vlad
Camelia
C
Tu
Tingting
T
Przybylski
Michael
M
D000595:Amino Acid Sequence; D055910:Antibodies, Immobilized / Q000737:chemistry / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D006801:Humans; D037102:Lectins; D046208:Microchip Analytical Procedures; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D037102:Lectins; D011506:Proteins; C002744:3-nitrotyrosine; D014443:Tyrosine