Affinity sample preparation

J. Gobom, M. Schuerenberg, M. Mueller, D. Theiss, H. Lehrach, and E. Nordhoff. a-Cyano-4-hydroxycinnamic Acid Affinity Sample Preparation. A Protocol for MALDI-MS Peptide Analysis in Proteomics. Anal. Chem., 73(2001) 434-438. 

Mueller, M., Theiss, D., Lehragh, H., Nordhoff, E. (2001). a-Cyano-4-hydroxycinnamic acid affinity sample preparation. A protocol for MALDI-MS peptide analysis in proteomics. Anal. Chem. 73, 434 38. 

The coupling of supercritical fluid extraction (SEE) with gas chromatography (SEE-GC) provides an excellent example of the application of multidimensional chromatography principles to a sample preparation method. In SEE, the analytical matrix is packed into an extraction vessel and a supercritical fluid, usually carbon dioxide, is passed through it. The analyte matrix may be viewed as the stationary phase, while the supercritical fluid can be viewed as the mobile phase. In order to obtain an effective extraction, the solubility of the analyte in the supercritical fluid mobile phase must be considered, along with its affinity to the matrix stationary phase. The effluent from the extraction is then collected and transferred to a gas chromatograph. In his comprehensive text, Taylor provides an excellent description of the principles and applications of SEE (44), while Pawliszyn presents a description of the supercritical fluid as the mobile phase in his development of a kinetic model for the extraction process (45). 

Probably the most common distillation method used as a form of sample preparation for chromatographic analysis is steam distillation [31,32]. Solvent extraction and gas phase stripping methods are generally inefficient procedures for isolating polar, acidic, or basic compounds in an aqueous matrix due to the low efficiency of water immiscible solvents for the extraction of these compounds and their low volatility and high water affinity which results in a very slow transfer to the gas phase using... 

As noted above, whole-cell MALDI-TOF MS was intended for rapid taxonomic identification of bacteria. Neither the analysis of specific targeted bacterial proteins, nor the discovery of new proteins, was envisioned as a routine application for which whole cells would be used. An unknown or target protein might not have the abundance or proton affinity to facilitate its detection from such a complex mixture containing literally thousands of other proteins. Thus, for many applications, the analysis of proteins from chromatographically separated fractions remains a more productive approach. From a historical perspective, whole-cell MALDI is a logical extension of MALDI analysis of isolated cellular proteins. After all, purified proteins can be obtained from bacteria after different levels of purification. Differences in method often reflect how much purification is done prior to analysis. With whole-cell MALDI the answer is literally none. Some methods attempt to combine the benefits of the rapid whole cell approach with a minimal level of sample preparation, often based on the analysis of crude fractions rather... 

The micropipette tip containing solid phases is a relatively new sample preparation technique that permits handling of microliter to submicroliter amounts of liquid samples, using the techniques of SPE, dialysis, and enzyme digestion. Various phases (reversed-phase, affinity, size-exclusion, etc.) are packed, embedded, or coated on the walls of pipette, permitting liquid samples to be transferred without undue pressure drop or plugging (Fig. 2.5). 

Surface enhanced laser desorption/ionization (SELDI) is a distinctive form of laser desorption ionization where the target plays an active role in the sample preparation procedure and ionization process [49]. Depending on the chemical or biochemical treatment, the SELDI surface acts as solid phase extraction or an affinity probe. Chromatographic surface is used for sample fractionation and purification of biological samples prior to direct analysis by laser desorption/ ionization. SELDI is mainly applied for protein profiling and in biomarker discovery by comparing protein profiles from control and patient groups. 

For example, our preliminary MALDI MS studies employed a somewhat unconventional sample preparation approach in which the protein deposition step preceded the matrix deposition step. (In the conventional approach, the protein is co-deposited with the MALDI matrix solution.). .. Thus, in the first/next/colleclively group of experiments, we will explore different sample preparation methods to determine if other protocols also lead to an inverse relationship between surface-protein binding affinity and the MALDI ion signal. 

SPME is a sample-preparation technique based on absorption that is useful for extraction and concentration of analytes either by submersion in a liquid phase or exposure to a gaseous phase (Belardi and Pawliszyn, 1989 Arthur et al., 1992). Following exposure of the fiber to the sample, absorbed analytes can be thermally desorbed in a conventional GC injection port. The fiber behaves as a liquid solvent that selectively extracts analytes, with more polar fibers having a greater affinity for polar analytes. Headspace extraction from equilibrium is based on partition coefficients of individual compounds between the food and headspace and between the headspace and the fiber coat-... 

Recently, in many demanding sample preparation situations, more selective sorbents have been used. Affinity type sorbents, particularly immunosorbents, have gained popularity in trace analysis, not only for biological macromolecules but also for small molecules, like aflatoxins. MIPs have properties resembling those of affinity phases and therefore they may find unique applications where other sample pretreatment methods are tedious. This relates both to the separation of a single analyte and to the separation of a group of related analytes from the sample matrix. 

Comparison of the profiles reveals that proteins present in the sample prepared by ammonium sulfate fractionation (Fig. 5.7A) were removed following affinity chromatography (Fig. 5.7B) and ion-exchange chromatography (Fig. 5.7C). The profile of the activity following ion-exchange HPLC (Fig. 5.7D) shows considerably less protein than was originally present. 

For the analysis of ribonucleosides the use of phenylboronate affinity gels is the most selective. If, however, in addition to ribonucleosides the investigator wishes to analyze for purine or pyrimidine bases, other methods of sample preparation must be investigated. 

The term solid-phase extraction was introduced by personnel of the J. T. Baker Company in 1982. The method consists of retention of the analytes from a liquid or gaseous sample to a solid stationary phase and subsequent removal of analytes using an appropriate eluent. The main purpose of SPE is isolation and preconcentration of compounds of interest or sample clean-up and simplification of the matrix. Application of this sample preparation technique also allows extract fractionation. As a result of significant reduction in the volume of organic solvents used, high recovery, and the possibility of process automation, SPE is a good alternative for conventional liquid-liquid extraction. According to their affinity for the compound of interest, stationary phases are classified as follows ... 

Improvements in sensitivity and LOD can have a profound effect on molecular biology applications. This will equate to reduced sample requirements, allowing for more assays to be done on small sample preparations and allowing precious samples to be better utilized. Improved sensitivity and LOD will also allow the use of antibodies with lower binding affinities and may allow for better quantitation of array data. 

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