This is a description of a comprehensive two-dimensional liquid chromatography (LC) system for the separation of protein mixtures. This system uses cation-exchange chromatography followed by reversed-phase chromatography (RPLC). The two LC systems are coupled by an eight-port valve equipped with two storage loops and under computer control. The RPLC effluent is sampled by both a UV detector and an electrospray mass spectrometer. In this way, complex mixtures of large biomolecules can be rapidly separated, desalted, and analyzed for molecular weight in less than 2 h. The system's utility is demonstrated with a mixture of standards and an Escherichia coli cell lysate.

Many G protein-coupled receptors (e.g. that of angiotensin II) activate phospholipase C␤, initially increasing intracellular calcium and activating protein kinase C. In the WB and GN4 rat liver epithelial cell lines, agonistinduced calcium signals also stimulate tyrosine phosphorylation and subsequently increase the activity of c-Jun N-terminal kinase (JNK). We have now purified the major calcium-dependent tyrosine kinase (CADTK), and by peptide and nucleic acid sequencing identified it as a rat homologue of human PYK2. CADTK/PYK2 is most closely related to p125 FAK and both enzymes are expressed in WB and GN4 cells. Angiotensin II, which only slightly increases p125 FAK tyrosine phosphorylation in GN4 cells, substantially increased CADTK tyrosine autophosphorylation and kinase activity. Agonists for other G protein-coupled receptors (e.g. LPA), or those increasing intracellular calcium (thapsigargin), also stimulated CADTK. In comparing the two rat liver cell lines, GN4 cells exhibited ϳ 5-fold greater angiotensin II-and thapsigargin-dependent CADTK activation thanWBcells.AlthoughmaximalJNKactivationbystressdependent pathways (e.g. UV and anisomycin) was equivalent in the two cell lines, calcium-dependent JNK activation was 5-fold greater in GN4, correlating with CADTK activation. In contrast to JNK, the thapsigargin-dependent calcium signal did not activate mitogen-activated protein kinase and Ang II-dependent mitogen-activated protein kinase activation was not correlated with CADTK activation. Finally, while some stress-dependent activators of the JNK pathway (NaCl and sorbitol) stimulated CADTK, others (anisomycin, UV, and TNF␣) did not. In summary, cells expressing CADTK/PYK2 appear to have two alternative JNK activation pathways: one stressactivated and the other calcium-dependent.

A two-dimensional liquid chromatography system is described here which uses size exclusion liquid chromatography (SEC) followed by reversed phase liquid chromatography (RPLC) to separate the mixture of peptides resulting from the enzymatic digestion of a protein. A novel LC/LC interface, using two RPLC columns in parallel rather than storage loops, joins the two chromatographic dimensions. This new interface design permits the use of conventional analytical diameter HPLC columns, 7.8 mm for SEC and 4.6 mm for RPLC, making construction and maintenance of this system very easy. The reversed phase chromatography utilizes 1.5 microns diameter, nonporous C-18 modified silica particles, which produce fast and efficient analyses. Following the high-resolution two-dimensional chromatographic separation, an electrospray mass spectrometer detects the peptide fragments. The mass spectrometer scans a 2000 m/z range to identify the analytes from their molecular weights. The analyses of tryptic digests of ovalbumin and serum albumin are each described.

The complete primary structure of the purple membrane protein bacteriorhodopsin, which contains 248 amino acid residues, has been determined. Methods used for separation of the hydrophobic fragments included gel permeation and reverse-phase high-pressure liquid chromatography in organic solvents. The amino acid sequence was determined by a combination of automatic Edman degradation and mass spectrometric methods. The total sequence was derived by ordering of the CNBr fragments on the basis of methionine-containing peptides identified by gas chromatographic mass spectrometry and by analysis of N-bromosuccinimide fragments containing overlaps between CNBr fragments. The present sequence differs from that recently reported by Ovchinnikov and coworkers with respect to an additional tryptophan (position 138) and several amino acid assignments.The purple membrane of a number of extremely halophilic bacteria-e.g., Halobacterium halobium-functions as a light-driven proton pump (1, 2). It contains a single protein, bacteriorhodopsin (Mr 26,000) with one molecule of retinaldehyde covalently bound to a lysine residue (3, 4). Bacteriorhodopsin forms a continuum of seven a helices, each of which spans the membrane and is largely embedded in it (5). Knowledge of the primary structure of bacteriorhodopsin is a prerequisite to studies of the mechanism of the proton pump and of the biogenesis of this interesting protein. In a recent paper, we reported (6) on the sequence of the first 102 amino acids and of 39 amino acids from the COOH terminus of this membrane protein. The present paper reports on its complete amino acid sequence (Fig. 1). Although no experimental details have appeared, the complete sequence has also been deduced by Ovchinnikov and coworkers (7,8), and partial sequences have been reported by other laboratories (3, 9, 10). The present sequence differs from that reported by Ovchinnikov and coworkers with respect to an additional tryptophan (position 138) and amino acid assignments at positions 105, 111, 117, 146, and 206. Of the total 248 amino acids present in bacteriorhodopsin, 70% are hydrophobic, and there is significant clustering of the hydrophobic as well as of the hydrophilic amino acids. MATERIALS AND METHODSMaterials. N-Bromosuccinimide (NBS), fluorescamine, and isothiocyanatophenylthiocarbamoylaminopropyl (IPTAP) glass were purchased from Pierce, and trypsin treated with L-(tosylamido-2-phenyl)ethyl chloromethyl ketone, clostripain, and elastase were from Worthington Biochemicals. ['4CISuc-cinic anhydride was from New England Nuclear. Tetraethyltetraamino (TETA) and aminoethylaminopropyl (AEAP) glass were gifts from Mark Horn of Sequemat, Watertown, MA. Other materials were as described previously.Preparation of Chymotryptic and CNBr Fragments. Purple membrane was isolated from H. halobium and apomembrane was prepared as described (6). Digestion of the apomembrane with chymotrypsin gave two fragments, C-1 and C-2, which were collected by centrifugation and separated on a Sephadex LH-60 column ...

Deuterium exchange of bovine cytochrome c has been monitored by electrospray ionization mass spectrometry. Different charge-state distributions in the mass spectrum appear to represent different protein conformations, but rapid interconversion of the conformations can lead to a coincidence of the deuterium exchange rates. When interconversion is blocked, the conformation corresponding to higher m/z (lower charge) exchanges more slowly, indicating a tightly folded state. Furthermore, the data suggest that at least two conformations can have identical charge-state distributions, but have different exchange rates. Thus, neither charge-state distribution nor deuterium exchange rate alone is a sufficient indicator of protein conformation.