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We report on the development of a parallel HPLC/MS purification system incorporating an indexed (i.e., multiplexed) ion source. In the method described, each of the flow streams from a parallel array of HPLC columns is directed toward the multiplexed (MUX) ion source and sampled in a time-dependent, parallel manner. A visual basic application has been developed and monitors in real-time the extracted ion current from each sprayer channel. Mass-directed fraction collection is initiated into a parallel array of fraction collectors specific for each of the spray channels. In the first embodiment of this technique, we report on a four-column semipreparative parallel LC/MS system incorporating MUX detection. In this parallel LC/MS application (in which sample loads between 1 and 10 mg on-column are typically made), no cross talk was observed. Ion signals from each of the channels were found reproducible over 192 injections, with interchannel signal variations between 11 and 17%. The visual basic fraction collection application permits preset individual start collection and end collection thresholds for each channel, thereby compensating for the slight variation in signal between sprayers. By incorporating postfraction collector UV detection, we have been able to optimize the valve-triggering delay time with precut transfer tubing between the mass spectrometer and fraction collectors and achieve recoveries greater than 80%. Examples of the MUX-guided, mass-directed fraction purification of both standards and real library reaction mixtures are presented within.

An automated parallel analytical/preparative HPLC/MS workstation has been developed to increase the analysis speed for characterizing and purifying combinatorial libraries. The system incorporates two columns operated in parallel for both LC/MS analysis and preparative LC/MS purifications (a technique we have named 'parallel Analyt/PrepLCMS'). Keys to the successful development of this system have been the design of a multiple sprayer IonSpray interface to support flows from multiple columns and the design of a valving configuration which is under complete software control for delivering the crude samples to the two HPLC columns from a single autosampler. In comparison with the conventional LC/MS systems, where compounds are analyzed sequentially (i.e., one at a time), our parallel Analyt/PrepLCMS system provides an important increase in sample throughput, especially critical in the field of combinatorial chemistry, where compound libraries are generated frequently in parallel arrays and require high-throughout analysis techniques. Minor hardware modifications to the HPLC system were made to facilitate parallel chromatography analyses. By incorporating fast chromatography and mass-based fraction collection on our new parallel Analyt/PrepLCMS system, we demonstrate that more than 200 compounds can be characterized per instrument per day, and more than 200 compounds can be purified per instrument per night. The concept, design, and applications of parallel HPLC/MS to combinatorial libraries are presented and discussed.

Rapid digestion of protein pp60(sup c-src) tyrosine kinase (TK) enables mapping of the time course for autophosphorylation of src TK on three tyrosine sites and provides direct evidence for an intramolecular pY530-SH2 complex. The digestion process involves perfusive-immobilized enzyme cartridges in combination with electrospray ionization mass spectrometry, and the results support earlier models on phosphorylation. The pY530-SH2 interaction modulates signal transduction through a five-fold down-regulation of src TK activity along with inhibition of protein-protein interactions.

The intracellular receptor autophosphorylation on tyrosine residues causes accumulation of different proteins with SH2 domains including c-src, during the signal transduction activated by epidermal growth factor. c-src and epidermal growth factor receptor (EGFR) kinase activities phosphorylate in vitro the C-terminal autophosphorylation domain of EGFR. The SH2 domain of c-src interacts with certain EGFR c-src phosphorylation sites. Such interaction is responsible for the regulation of growth factor receptor activity and signal transduction.

The forward and reverse pp60(super c-src) tyrosine kinase (srcTK) reactions are responsible for controlling the intracellular levels of protein tyrosine kinase phosphorylation. A study conducted on the phosphorylation and dephosphorylation reactions indicate that the enzyme catalyzes the phosphorylation of peptides that bind to its SH2 region. SrcTK is responsible for dephosphorylation of peptides in the presence of MgATP which is hydrolyzed to MgADP. Substances that suppress the ATP binding act as inhibitors of dephosphorylation.

Autophosphorylation of pp60(super c-src) tyrosine kinase (srcTK) is an intermolecular process. The electrospray ionization mass spectrum of srcTK reveals that MgATP is effective in increasing the phosphorylation process. The enzyme srcTK is similar to receptor tyrosine kinases in its ability to dimerize and indicates that the dimerization which precedes activation does not necessitate binding to a membrane or a hydrophobic N-terminal region.

Reports on the rapid digestion of tyrosine kinase in combination with electrospray ionization mass spectrometry. Autophosphorylation of three tyrosine sites; Result of surface plasmon resonance detection and size exclusion chromatography; Retention of kinase activity.