Pau-Miau Yuan

Homogeneous Cell- and Bead-Based Assays for High Throughput Screening Using Fluorometric Microvolume Assay Technology.

High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds has resulted in a requirement for assays and instrumentation that are amenable to nonradioactive formats and that can be miniaturized. Homogeneous assays that minimize upstream automation of the individual assays are also preferable. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based platform for the development of nonradioactive cell- and bead-based assays for HTS. This technology is plate format-independent, and while it was designed specifically for homogeneous ligand binding and immunological assays, it is amenable to any assay utilizing a fluorescent cell or bead. The instrument fits on a standard laboratory bench and consists of a laser scanner that generates a 1 mm2 digitized image of a 100-μm deep section of the bottom of a microwell plate. The instrument is directly compatible with a Zymark Twister™ (Zymark Corp., Hopkinton, MA) for robotic loading of the scanner and unattended operation in HTS mode. Fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using data processing. Unbound flurophore comprising the background signal is ignored, allowing for the development of a wide variety of homogeneous assays. The use of FMAT for peptide ligand binding assays, immunofluorescence, apoptosis and cytotoxicity, and bead-based immunocapture assays is described here, along with a general overview of the instrument and software.

Structural determination of the essential serine and glycosylation sites of carboxypeptidase P.

Through a series of kinetic studies involving the inactivation effects of diisopropylfluorophosphate, an affinity label that modifies the active site serine residue involved in the mechanism of action, it has been firmly established that carboxypeptidase P (CPP) requires a serine residue for catalytic activity. The essential kinetic parameters were determined to be 1.33 mM for the apparent dissociation constant with a limiting half-life of inactivation of 20.1 min. Structural elucidation of the primary amino acid sequence surrounding the essential serine, and comparing that with the reactive site of carboxypeptidase Y (CPY), revealed a significant degree of homology at the active site between these two enzymes. These regions, however, were quite divergent from other known serine proteases, leading to the speculation that these serine exopeptidases may comprise a unique family in the overall classification of serine proteases. It was established that CPY could be inactivated with either of the classic histidine affinity labels tosylphenylalanylchloromethyl ketone (TPCK) or carbobenzoxyphenylalanylchloromethyl ketone (ZPCK) with Kis of 1.2 and 12.8 microM, respectively. This is in marked contrast to CPP, which was unaffected by saturating levels of the known histidine affinity labels, TPCK, tosyllysylchloromethyl ketone, or ZPCK. This point may be a significant element in differentiating specificity among these two serine proteases. Further investigation into the structural nature of CPP revealed that it is a glycoprotein with a single site of carbohydrate attachment. In addition, the carbohydrate moiety itself appears to contribute 1217 Da to the overall molecular weight and it is characterized as an asparagine linked high mannose type. This is significantly different from CPY with its four sites of carbohydrate attachment contributing approximately 17% to its molecular weight.

A Practical Approach for Isolation and Characterization of Glycosylation Sites of Glycoproteins Bearing N- and/or O-Linked Carbohydrate Chains

Publisher Summary This chapter presents a practical approach for isolation and characterization of glycosylation sites of glycoproteins bearing N- and/or O-linked carbohydrate chains. Two glycoproteins were chosen as model glycoproteins for this study, namely, tissue plasminogen activator (t-PA) and human chorionic gonadotropin (HCG). t-PA bears three different N-linked carbohydrate chains, and hCG has several N-linked and O-linked carbohydrate chains attached. Chemicals were either reagent or HPLC grade, as appropriate. Sequence analysis was performed either on the ABI Model 477A or 473A Sequencer using standard cycles. Mass analysis was carried out on a prototype matrix-assisted laser desorption time-of-flight mass spectrometer. Peptides were separated on an ABI Model 172 HPLC System that consisted of a 140B Solvent Delivery System, a 785A Programmable Absorbance Detector, and a 112A Injector, using a solid-particle, nonporous column.

Obtaining Primary Sequence Information from the Carboxy Terminal End of Polypeptides

The principles of end labeling have been firmly established in the DNA sequencing field (for example, Smith et. al., 1986). In fact, end labelling in conjunction with an appropriate separation technique is the cornerstone of this technology. Unfortunately, the intrinsically higher chemical diversity of proteins built from 20+ different amino acids (compared with the 4 bases in DNA) with a wide range of chemical reactivity has impeded the development of such techniques for protein chemists. Such procedures would be useful in both finding the C-terminal fragment for sequence analysis, and making alignments from partial cleavage maps. Selective isolation followed by amino terminal sequencing is an alternative to direct chemical sequencing from the carboxy terminal.

A strategy of obtain internal sequence information from blotted proteins after initial N-terminal sequencing

Publisher Summary To generate internal peptide fragments for the identification of sequences from N-terminally blocked proteins, or for the maximization of sequence, information from larger proteins require purification of additional protein sample. With the advent of high sensitivity sample preparation systems employing capillary HPLC, it has become feasible to explore the generation and purification of internal peptide fragments from modest amounts of protein (60 picomole) immobilized onto polyvinylidene difluoride (PVDF) membrane that have previously been subjected to Edman degradation. Initial investigations reveal that after proteins have subjected to Edman chemistry, they are refractory to digestion by the enzymes trypsin, Lys-C, and Glu-C. It is possible to generate internal fragments using chymotrypsin, but the extensive auto-digestion products contaminate the subsequent peptide maps. Chemical cleavage methods were employed resulting in great success. Two proteins—that is, carbonic anhydrase and transferrin, are chosen as models for this study. The experiments discussed in this chapter demonstrates the generation, extraction, and the subsequent purification strategy of internal fragments using both cyanogen bromide to cleave proteins at methionine, and incubation in formic acid at elevated temperature to cut between the aspartic acid and proline. There are two advantages to performing cyanogen bromide digestions in 70% formic acid at an elevated temperature: first, the methionine specific cleavage occurred faster, and second, the cleavage between aspartic acid and proline pairs were catalyzed. This resulted in the generation of more peptide fragments for all samples tested in a relatively short time.

A Unified Approach to Glycoprotein Primary Structure Analysis: Identification, Isolation, and Characterization of both Peptide and Pendant Carbohydrate of Glycopeptides

Publisher Summary This chapter outlines the general strategy for the analysis of glycoproteins, utilizing lectin staining, available peptide separation and sequencing techniques, a simple micro-batch affinity method, PDMS, and PMP labeling of carbohydrates. A classical approach of carbohydrate chemists to glycoprotein analysis is to completely deglycosylate the glycoprotein (using hydrazinolysis), isolate and purify the resulting oligosaccharides, and then do structure determinations. Disadvantages to this scheme are that site information is lost and the peptide bonds are usually completely destroyed as well. Advantages are that most protein laboratories have the necessary instrumentation, and both peptide and carbohydrate structural information can be acquired at levels commensurate with the demands of modern protein chemistry.

Studies on C-Terminal Analysis

Despite the fact that end labelling has been widely used for DNA analyses, and in combination with an appropriate separation technique is central to all methods for DNA sequencing (1), reports of end-labelling of proteins are quite rare. The intrinsically higher chemical diversity of proteins (twenty different amino acids compared with the four bases in DNA) with a wide range of chemical reactivity has impeded the development of such techniques for protein chemists. Such procedures would be useful in both finding the C-terminal fragment for amino terminal sequence analysis, as well as making alignments from partial cleavage maps. This could increase the analytical efficiency of sequencing projects tremendously by allowing workers to concentrate on peptides of particular interest. Selective isolation of the C-terminal peptide followed by amino terminal sequencing is also a potential alternative to direct chemical sequencing (2) from the carboxy terminus as a method for obtaining C-terminal sequence information.