Mass spectrometry affinity

Affinity Capillary Electrophoresis-Mass Spectrometry. Affinity capillary electrophoresis was originally used for the determination of the binding constants of small molecules to proteins (47-49). This solution-based technique is rapid and requires only small amounts of ligands. Affinity constants are measured based on the mobility change of the ligand on interaction with the receptor present in the electrophoretic buffer (50). By combining affinity capillary electrophoresis with on-line mass spectrometric detection and... 

Zn Not reported Not reported Binding detected by mass spectrometry, affinity columns and assays [23, 27]... 

This technique provides quantitative information about tautomeric equilibria in the gas phase. The results are often complementary to those obtained by mass spectrometry (Section VII,E). In principle, gas-phase proton affinities, as determined by ICR, should provide quantitative data on tautomeric equilibria. The problem is the need to correct the measured values for the model compounds, generally methyl derivatives, by the so-called N-, 0-, or S-methylation effect. Since the difference in stability between tautomers is generally not too large (otherwise determination of the most stable tautomer is trivial) and since the methylation effects are difficult to calculate, the result is that proton affinity measurements allow only semi-quantitative estimates of individual tautomer stabilities. This is a problem similar to but more severe than that encountered in the method using solution basicities (76AHCS1, p. 20). 

Crude chloroform-methanol-water (30 60 8, v/v) extracts of immunostainedTLC bands were analyzed without further purification by nanoelectrospray low-energy mass spectrometry. The authors showed that this effective PLC/MS-joined procedure offers a wide range of applications for any carbohydrate-binding agents such as bacterial toxins, plant lectins, and others. Phenyl-boronic acid (PBA) immobilized on stationary support phases can be put to similar applications. This technology, named boronate affinity chromatography (BAC), consists of a chemical reaction of 1,2- and 1,3-diols with the bonded-phase PBA to form a stable... 

Li, S. and Dass, C., Iron(III)-Immobilized Metal Ion Affinity Chromatography and Mass Spectrometry for the Purification and Characterization of Synthetic Phosphopeptides, Anal. Biochem., 270, 9, 1999. 

Another means of moving beyond pure protein preparations to high-throughput characterization of proteomes is to enrich for phosphopeptides from complex mixtures by metal affinity chromatography (Andersson and Porath, 1986). Using this method, protein mixtures are proteolyzed to create peptides and phosphorylated peptides are enriched by metal affinity chromatography and subsequently identified by mass spectrometry. This method is limited, however, because in many cases phosphopeptides absorb poorly or nonphosphorylated peptides absorb nonspecifically to the metal affinity resins (Ahn and Resing, 2001). 

Figure 2.7. Identification ofphosphoproteins by site-specific chemical modification. A. Method of Zhou et al. (2001) involves trypsin digest of complex protein mixture followed by addition of sulfhydryl groups specifically to phosphopeptides. The sulfhydryl group allows capture of the peptide on a bead. Elution of the peptides restores the phosphate and the resulting phosphopeptide is analyzed by tandem mass spectrometry. B. Method of creates a biotin tag in place of the phosphate group. The biotin tag is used for subsequent affinity purification. The purified proteins are proteolyzed and identified by mass spectrometry.

The second method also relies on site-specific chemical modification ofphosphoproteins (Oda et al., 2001). It involves the chemical replacement of phosphates on serine and threonine residues with a biotin affinity tag (Fig. 2.7B). The replacement reaction takes advantage of the fact that the phosphate moiety on phosphoserine and phosphothreonine undergoes -elimination under alkaline conditions to form a group that reacts with nucleophiles such as ethanedithiol. The resulting free sulfydryls can then be coupled to biotin to create the affinity tag (Oda et al., 2001). The biotin tag is used to purify the proteins subsequent to proteolytic digestion. The biotinylated peptides are isolated by an additional affinity purification step and are then analyzed by mass spectrometry (Oda et al., 2001). This method was also tested with phosphorylated (Teasein and shown to efficiently enrich phosphopeptides. In addition, the method was used on a crude protein lysate from yeast and phosphorylated ovalbumin was detected. Thus, as with the method of Zhou et al. (2001), additional fractionation steps will be required to detect low abundance phosphoproteins. 

Figure 5.11. Generic approaches to identify interacting proteins within complexes. The complex is isolated from cells by affinity purification using a tag sequence attached to a protein known to be in the complex. Alternatively, the complex can be immunprecipitated with an antibody to one of the proteins in the complex. The proteins are resolved by polyacrylamide gel electrophoresis, proteolyzed, and the mass of the resulting peptides is determined by mass spectrometry. Alternatively, the proteins can be proteolyzed and the resulting peptides resolved by liquid chromatography. The peptide masses are then determined by mass spectrometry and used for database searching to identify the component proteins.

The difficulty with protein arrays is that proteins do not behave as uniformly as nucleic acid. Protein function is dependent on a precise, and fragile, three-dimensional structure that may be difficult to maintain in an array format. In addition, the strength and stability of interactions between proteins are not nearly as standardized as nucleic acid hybridization. Each protein-protein interaction is unique and could assume a wide range of affinities. Currently, protein expression mapping is performed almost exclusively by two-dimensional electrophoresis and mass spectrometry. The development of protein arrays, however, could provide another powerful... 

Von Haller, P.D., Yi, E., Donohoe, S., Vaughn, K., Keller, A., Nesvizhskii, A.I., Eng, J., Li, X.J., Goodlett, D.R., Aebersold, R., Watts, J.D. (2003). The Application of New Software Tools to Quantitative Protein Profiling Via Isotope-coded Affinity Tag (ICAT) and Tandem Mass Spectrometry II. Evaluation of Tandem Mass Spectrometry Methodologies for Large-Scale Protein Analysis, and the Application of Statistical Tools for Data Analysis and Interpretation. Mol. Cell. Proteomics 2, 428 -42. 

Bouchonnet, S., and Y. Hoppiliard. 1992. Proton and Sodium Ion Affinities of Glycine and Its Sodium Salt in the Gas Phase. Ab Initio Calculations. Qrg. Mass Spectrometry 27, 71-76. 

It should be emphasized that the nature of all presented protocols is very general and, thus, their application for a comprehensive characterization of your favorite multiprotein complex (YFMPC) in yeast might require only minor modifications. The logical sequence of all required steps is schematically shown in Fig. 2.1. The initial large-scale Ni affinity isolation of eIF3 followed by mass spectrometry (MS) of its subunit composition has already been described (Asano et al, 2002), and methods for identification of protein-protein interactions such as yeast two-hybrid (Y2H) and in vitro glutathione-S-transferase (GST) pull-down analysis are presented in volume 429. This chapter focuses on a description of the small-scale one-step in vivo affinity purification techniques that were used to determine the effects of deletions and... 

Fetsch PA, Simone NL, Bryant-Greenwood PK, et al. Proteomic evaluation of archival cytologic material using SELDI affinity mass spectrometry potential for diagnostic applications. Am. J. Clin. Pathol. 2002 118 870-876. 

Guerrero C, Tagwerker C, Kaiser P, et al. An integrated mass spectrometry-based proteomic approach quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26 S proteasome-interacting network. Mol. Cell. Proteomics. 2006 5 366-378. 

---