On-line bioaffinity-electrospray mass spectrometry for simultaneous detection, identification, and quantification of protein-ligand interactions

Abstract

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 ␤-amyloid (A␤) epitope peptides bound to anti-A␤ 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-␣-synuclein-human ␣-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. (J Am Soc Mass Spectrom 2010, 21, 1643-1648 [3,4]. In particular, SPR has developed into an efficient tool for analysis of biomolecular recognition processes at a biosensor surface, and has been applied to the quantification of a variety of biopolymer interactions [4,5]. A recently-explored alternative to SPR is the surface acoustic wave (SAW) technology in which the piezoelectric effect of mass differences is employed for bioaffinity detection [6 -9]. SAW is now becoming increasingly important for the study of biomacromolecular interactions due to its high detection sensitivity in dilute solutions.Advantages of SAW in comparison to classical immuno-analytical techniques are the direct and rapid determination of association/dissociation constants with small sample amounts, and without labeling approaches or recalibration for buffer changes being required [6]. While providing sensitive and accurate determinations of binding/dissociation constants (K i or K D ), a major limitation of all bioaffinity methods is the lack of direct identification of the affinity-bound ligands. In contrast, the combination of biosensor detection and mass spectrometry enables both identification and quantification of bioaffinity interactions of biopolymers. Here we describe an on-line combination of an SAW biosensor with electrospray ionization mass spectrometry, SAW-ESI-MS. The on-line coupling between the SAW-sensor chip and the ESI-MS source was achieved by a incorporating a standard trapping column setup, using a six-port valve, a guard column, and a micropump system. The six-port valve interface provides both ligand concentration and an...

Identification and Affinity-Quantification of ß-Amyloid and α-Synuclein Polypeptides Using On-Line SAW-Biosensor-Mass Spectrometry

“…The chip surface was prepared and cleaned before binding and affinity determinations by 45 min washing with a 1:1 mixture (v/v) of concentrated sulphuric acid and hydrogen peroxide (30%). Immobilization of proteins was performed by covalent binding of a monolayer (SAM) of 16-mercaptohexadecanoic acid as previously described [13].…”

“…We have developed a new interface for on-line bioaffinity-mass spectrometry coupling of a surface acoustic wave (SAW) biosensor to electrospray ionization mass spectrometry (SAW-MS) [13], which provides both affinity detection and quantification and the mass spectrometric chemical structure analysis of ligands. Key element of the on-line biosensor-MS is a microfluidic interface enabling affinity-enrichment, desalting of samples from the biosensor, and transfer of ligands to the ESI-MS source.…”

When is Mass Spectrometry Combined with Affinity Approaches Essential? A Case Study of Tyrosine Nitration in Proteins

“…N-α-Fmoc protected amino acids attached to NovaSyn TGA resin was from NovaBiochem (Laufelfingen, Switzerland). A K5-Ssens Biosensor (SAW-Instruments, Bonn, Germany) was used for online bioaffinity-MS with an interface developed in our laboratory, as previously described [50]. The antibody was immobilized on a gold chip, following formation of a self-assembled monolayer (SAM) surface of 16-mercaptohexadecanoic acid for 12 h at 25°C.…”

“…Following ligand association, elution was carried out with glycine buffer, and removal of salts was performed by washing with 0.3 % HCOOH. Elution and transfer into the ESI source of a Bruker Esquire 3000+ mass spectrometer was performed as previously described [50]. Determination of dissociation constants was performed with 150 μL (300 nM) immobilized antibody at a 20 μL min -1 flow rate in PBS buffer, pH 7.5, followed by regeneration with 150 μL glycine buffer, pH 2.…”

“…A direct online combination of bioaffinity and mass spectrometry [50], a new method recently developed in our laboratory was employed for the simultaneous characterization and quantification of the interaction of tyrosine-nitrated peptides with the anti-3NT-antibody. The online affinity-MS approach utilized a surface-acoustic wave (SAW) biosensor [51], with the anti-3NT antibody immobilized on the biosensor chip surface via a self-assembled monolayer.…”