Mass spectrometry probe affinity

An obvious next step is the combination of the two techniques, BIA/MS with bioreactive mass spectrometry probe tips. Such an approach would thereby allow (all on a single surface), the real-time observance of affinity interaction followed by enzymatic modification and mass spectrometric characterization of retained ligands. 

Yip, T-T. Hutchens, T.W. Affinity Mass Spectrometry. Probes with Surfaces Enhanced for Affinity Capture (SEAC) of Lactoferrin, Exp. Biol. Med. 28, 39-58 (1997). 

The techniques developed to study protein interactions can be divided into a number of major categories (Table 31.1), including bioconjugation, protein interaction mapping, affinity capture, two-hybrid techniques, protein probing, and instrumental analysis (i.e., NMR, crystallography, mass spectrometry, and surface plasmon resonance). Many of these methods are dependent on the use of an initial bioconjugation step to discern key information on protein interaction partners. 

Recent achievements in the development of active-site directed affinity probes for proteases and other enzyme classes provide direct chemical labeling of proteases of interest in the biological system (24-27). These specific activity probes allow joint evaluation of selective protease inhibition concomitant with labeling of relevant protease enzymes for more analyses. Moreover, activity-based probes that selectively label the main protease subclasses—cysteine, serine, metallo, aspartic, and threonine—can provide advantageous chemical approaches for functional protease identification. Activity probe labeling of proteases allows direct identihcation of enzyme proteins by tandem mass spectrometry. Such chemical probes directed to cysteine proteases have been instrumental for identification of the new cathepsin L cysteine protease pathway for neuropeptide biosynthesis, as summarized in this article. 

Finally, using PNA as an affinity capture reagent recently was extended to probing RNA-protein complexes (RNPs) in cells (33). In this application, the PNA is functionalized with a peptide that allows uptake into cells and is complementary to an RNA component of an RNP. The PNA also bears two affinity tags, the first of which is a benzophenone-modifled phenylalanine residue that can photocross-link the PNA to a protein present in the RNP. The second tag is a biotin group, which allows the purification of the cross-linked PNA-protein. Subsequent analysis by mass spectrometry identifies both the protein and its cross-linking site. As is the case for PNA used to deliver a fluorophore to a specific site in an RNA, this method requires that the PNA not disrupt the structure being probed. 

We have developed an analogous, but more robust system which is not necessarily constraint by the aforementioned limitations. The obvious extension has been to couple an affinity-based separation with mass spectrometry. Hutchens et al. have shown that affinity probe surfaces can be ust to capture specific protein ligands allowing detection by laser desorption mass spectrometry (. The limitations to their technique have been that the surface area for ligand capture is quite small and salt (or detergent) contaminants are still problematic. Perfusive affinity resins, on the other hand, provide a tremendous surface area for binding. The nature and composition of the solvents required for affinity chromatography, however, are not directly compatible with mass spectrometric analysis. 

D. Surface Enhanced Affinity Capture and Probe Affinity Mass Spectrometry... 

AH Brockman, R Orlando. New immobilization chemistry for probe affinity mass spectrometry. Rapid Commun Mass Spectrom 10 1688-1692, 1996. 

Organophosphorus Chemistry series regularly lists new mass spectra in the Physical Methods chapter . With this in mind, an approach which considers fundamental aspects of organophosphorus ions (i.e. structure and reactivity) in the gas phase has been adopted. The gas-phase structure and reactivity of ions can be probed via several different techniques, including thermochemical measurements, kinetic energy release of metastable ions, collisional activation mass spectrometry, neutralization reionization mass spectrometry and ion-molecule reactions. An example is the molecule HCP (Table 1) its ionization potentiaP, proton affinity and the IR and rotational spectroscopy of the HCP ion " have all been determined in the gas phase. Another important tool for understanding the structure and reactivity of gas phase ions is ab initio molecular orbital theory. With advances in computational hardware and software, it is now possible to carry out high-level ab initio calculations on smaller systems. Indeed, the interplay between experiment and theory has fuelled many studies ... 

Aptamers hold great promise as molecular recognition tools for their incorporation into analytical devices, and in particular, they can be used as immobilized ligands in separation technologies (Clark and Remcho, 2002), as affinity probes in capillary electrophoresis-based quantitative assays (Kotia and McGown, 2000), in affinity matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)(Dick and McGown, 2004), and as biocomponents in biosensors (Tombelli et al., 2005). Other aptamer-based bioanalytical applications have been reviewed (Yon et al., 2003 Mukhopadhyay, 2005). 

Chen, C.T. and Chen, Y.-C. (2005) Fe304/Ti02 core/shell nanoparticles as affinity probes for the analysis of phosphopeptides using Ti02 surface-assisted laser desorption/ionization mass spectrometry. Anal. Chem., 77, 5912-5919. 

Chen, W.-Y, Chen, Y.-C. (2006) Affinity Based Mass Spectrometry by Using Iron Oxide Magnetic Particles as the Matrix and Concentrating Probes for SALDI MS Analysis of Peptides and Proteins. Anal. Bioanal. Chem. 386 699-704. 

In 1993, Hutchens and co-workers described surface-enhanced laser desorption/ionization (SELDI) technique, an affinity technology, which has progressed over the last decade to become a powerful analytical, an on-plate approach (Hutchens and Yip 1993). SELDI is a distinctive form of laser desorption/ionization (LDI) mass spectrometry in which the EDI probe plays an active role in the homogenization, preconcentration, amplification, purification, extraction, enrichment digestion, derivatization, synthesis, separation, and detection with complementary techniques, prior to the desorption and ionization of the analytes by MALDI (Merchant and Weinberger 2000). The principle of this approach is very simple. Biomolecules are captured by adsorption, partition, electrostatic interaction, or affinity chromatography on a solid-phase protein chip surface. Although SELDI provides a unique sample preparation platform, it is similar to MALDI-MS in that a laser... 

F. 10 Schematic representation for the photo-affinity-based ABPP strategy. The structure of HDAC photo-crosslinking probe (SAHA-BPyne) is shown in the middle [115]. ABPP activity-based protein profiling, HDAC histone deacetylase, SAHA suberoylanilide hydroxamic acid, CuAAC copper(l)-catalyzed azide-alkyne cycloaddition, LC-MS/MS liquid chromatography-tandem mass spectrometry... 

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