Peer Reviewed: Biomolecular Interaction Analysis Mass Spectrometry

BIA/MS can detect and characterize proteins in complex biological fluids at the low- to ... SPRi-MALDI MS: characterization and identification of a ki...
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BIA/MS can detect and characterize proteins in complex biological fluids at the low- to subfemtomole level. s the result of worldwide efforts, the genomes of several organisms have been or will be completely sequenced (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/Entrez/ Genome/org.html). Completed genomes are being cataloged into databases, which represent libraries that can be translated into all the potential proteins contained within the respective organism. Thus, genome databases will become the foundation for the much broader field of proteomics, wherein the structure and function of specific proteins are investigated. These studies are intrinsically complicated, presenting multidimensional problems. To begin with, a protein of interest generally resides in a complex biological system and often constitutes only a small fraction of the system’s total protein content. Therefore, the protein must be fractionated from the bulk endogenous compounds and the structure characterized. Proteome investigation then takes on the new dimension of protein function, emphasizing quaternary structure (polypeptide complexes resulting in functional proteins) and the biomolecular interaction partners (receptor–ligand interactions). Finally, it is often necessary to quantitatively

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monitor expression profiles to understand protein function as part of a complete cellular system. Proteome investigations will demand much of the analytical sciences and its instrumentation. Thus, there exists a growing need for concerted, multi-instrument approaches capable of highly sensitive analyte fractionation and protein structure and function characterization. Two techniques, surface plasmon resonance biomolecular interaction analysis (SPR-BIA) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS have risen over the last decade to the forefront of protein characterization. SPR-BIA is being used to study protein function by monitoring the biomolecular interactions between a solution-phase analyte and a biomolecular complement immobilized to the surface of an SPRactive sensor chip. MALDI-TOF MS is being used in numerous structural analyses, including the identification of proteins and their post-translational modifications. An additional benefit of SPR-BIA is that it can be used to isolate proteins from solution based on a form of planargeometry affinity chromatography. The isolated proteins can be analyzed directly from the sensor chip using

MALDI-TOF MS. Thus, SPR-BIA and MALDI-TOF MS serve as enabling technologies for a more concerted analysis termed biomolecular interaction analysis MS (BIA/MS). Over the past few years, we have been actively developing BIA/MS (1–7) using the approach depicted in Figure 1. In this report, we describe the root techniques of BIA/MS, the interface between them, past uses, and an approach to analyzing proteins directly from natural biological systems.

SPR-BIA Biosensors using SPR detection are now commercially available (8, 9). With the most popular chip-based instruments, SPR-BIA technology is used primarily for realtime investigation of biomolecular recognition events. Briefly stated, a sensor chip surface, comprised of an affinity receptor-derivatized gold layer on a glass substrate, is monitored using SPR while the chip surface is exposed to a complementary affinity ligand. Differences in surface concentration arising from receptor–ligand interactions result in a refractive index change at the gold/receptor interface. This, in turn, varies the resonance angle at which light is absorbed into the surface interface. With proper calibration, changes in the SPR resonance angle can be equated to the amount of material retained on the sensor chip surface. The sensor chip and SPR detector are combined with a microfluidics system that can route fluids over the chip’s surface. In doing so, multiple sites on the chip can be addressed for various purposes while each site is monitored simultaneously by SPR. Several excellent reviews are available explaining the concepts and applications of SPRBIA (10–13). Determining the affinity constants of biomolecular interactions is one of the most common uses of SPRbased biosensors (14). The SPR data—in the form of a

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relatively large changes at both the injection front and end because of the bulk refractive index changes between the continuousflow buffer and the analytical solution and the biomolecular interaction between calmodulin and melittin. Data in the association and dissociation regions are exclusively caused by the interaction between calmodulin and melittin. This data can be curve-fit according to various interaction models to determine association and dissociation rates and subsequently the dissociation constant.

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Constant improvements in instrumentation, functional matrixes, and sample preparation methods have made Receptor immobilization SPR-BIA Ligand binding MALDI-TOF MS one of 80000 FC1 the most practiced forms 70000 FC1 1500 FC2 FC2 60000 of biological MS. Several 50000 excellent reviews are avail1000 , immobilization , Binding 40000 able explaining the con30000 cepts and applications of 500 20000 MALDI-TOF MS (15–19). 10000 Binding immobilization MALDI-TOF MS possesses 0 0 the virtues of high speed 0 500 1000 1500 5000 5500 6000 6500 and sensitivity, detects all Time (s) Time (s) the ions in a single desorpFIGURE 1. BIA/MS. tion/ionization event (no scanning), and offers a mass Two-analyte BIA/MS assay for toxic-shock syndrome toxin-1 (TSST-1, purple circles) and staphylococcal enterotoxin B (SEB, blue squares). Flow cells (FC) are derivatized with either anti-TSST-1 IgG ( ) in FC1 or a combination of anti-TSST-1 and anti-SEB ( ) range of 1–1,000,000 Da. IgG in FC2. A sample containing both TSST-1 and SEB (in a large excess of albumin carrier) was flowed serially over both flow The technique also provides cells and the binding was monitored with SPR (for proteins, 1000 RU = 1 ng material per flow cell). MALDI-TOF MS spectra the equally important simtaken directly from the flow cells show retention of TSST-1 in FC1 and both TSST-1 and SEB in FC2. ultaneous mass analysis of complex mixtures, such as proteolytic digests and biological fluids. However, it is sensorgram that reports response versus time—are used to generally known that during direct analyses of complex determine the kinetics (on/off rates) of the biomolecular biological mixtures, specific peptides or proteins of interest interaction. The on/off rates can then be used to calculate may be obscured in the analysis because of the complexity the dissociation constant. of the fluid (as discussed later). Fractionation, with varying Sensorgrams reporting the interactions between melittin degrees of specificity, is therefore necessary to isolate choimmobilized to the sensor chip surface and solutions containing varying concentrations of calmodulin are shown in sen analytes from solution for a more thorough MALDIFigure 2. Four distinct regions are observed in the sensorTOF MS analysis. In this regard, SPR-BIA serves as an grams: injection front, association while exposed to analyte, ideal means of fractionating analytes before MALDI-TOF injection end, and dissociation. The response curves exhibit MS analysis.

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BIA/MS In its most basic form, BIA/MS is used typically to determine the affinity constant(s) of a biomolecular interaction, confirm that only the expected ligands participate in the interaction, and derive the dissociation constant (6). MALDI-TOF MS is used to confirm that the results are only due to the correct molecular mass version of the ligand. When the molecular mass is not the one expected, MALDI-TOF MS is used to determine whether the unknown components are retained through a nonspecific interaction with the sensor chip or through a specific interaction with the immobilized receptor. In the latter case, MALDI-TOF MS can recognize variants of the targeted ligand because the signals in the spectra are massshifted due to point mutations or chemical modification. These variants can compete with the wild-type ligand for the receptor (6). Such information is of great importance because the dissociation constants span many decades in strength and because ligand variants present even at trace levels in solution can contribute significantly to a binding curve. If multiple ligands compete for an immobilized receptor, MALDI-TOF MS data can assist in the curve-fitting of SPR-BIA data by determining the number of components bound to a receptor and their respective molecular masses. The analysis can be further enhanced when semiquantitative MALDI-TOF MS (20) is used to produce separate binding curves for each ligand, the sum of which adds to the composite binding curve recorded during SPR-BIA (6).

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Although SPR-BIA followed by MALDI-TOF MS is a simple concept, achieving the high sensitivity (detection at a