Fast characterization of intact proteins using a high-throughput eight-channel parallel liquid chromatography/mass spectrometry system.

The preparation of protein substrates requires that a large number of chromatographic fractions be analyzed for the presence of reactants, products and by-products. Analyses using linear matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) or single column liquid chromatography/mass spectrometry (LC/MS) have been inadequate because of mass resolution or throughput. Therefore, a high-throughput method employing an eight-channel parallel reverse-phase LC/MS system was developed. This system is capable of screening fractions from preparative ion-exchange chromatography with the required mass accuracy and throughput so that the protein purification process can be monitored in a relatively short period of time. As an example, the purification and analysis of an acylated protein with a molecular weight of 8.9 kDa is described and the detection of a contaminating by-product that differs in size by less than 20 Da is demonstrated. Using the current instrumentation and approach, it is practical to analyze 50 protein-containing fractions from column chromatography in less than 1 hour using parallel LC/MS.

An integrated ten-pump, eight-channel parallel LC/MS system for automated high-throughput analysis of proteins.

An integrated 10-pump eight-channel LC/MS system has been developed for automated high-throughput analysis of intact proteins in recombinant protein purification processes. The key features of the system include (1) a compact 10-pump HPLC module that uses two pumps to generate a binary gradient and 8 pumps to deliver the mixed gradient to eight independent flow channels; (2) a TOF mass spectrometer with an eight-channel multiplexed ESI interface, which records separate data for all eight channels over each HPLC run cycle; and (3) highly automated data processing software that allows unattended calculation of protein molecular weight (in Da) from original mass spectral data (in m/z). This system was used in the routine screening of fractions from preparative scale chromatography to monitor the purification process with the required mass accuracy and throughput. As an example, the production and purification of an acylated protein with a molecular weight of 9 kDa is described. Using this off-line approach, it is practical to fully characterize protein-containing fractions from column chromatography with an overall analytical throughput of 1 min/protein sample with minimum operator involvement.

Expression of PEP-19 inhibits apoptosis in PC12 cells.

PEP-19 is a calmodulin-regulatory protein found specifically within neurons, though cellular functions of this protein have not been determined. In an effort to define potential effects of PEP-19, PC12 cell lines expressing this protein were generated and subjected to apoptotic stimuli. As measured by LDH release, cell death in PEP-19 expressing cells was 2- to 5-fold less following u.v. irradiation, and 2- to 4-fold less following staurosporine treatment than controls. Additionally, PEP-19-expressing cells displayed decreased DNA ladder formation, chromatin and condensation, caspase activation following staurosporine treatment. Overall, these results demonstrate that PEP-19 can inhibit apoptotic processes in PC12 cells, suggesting a potential regulatory mechanism for pathways leading to cell death.

Small proteins that modulate calmodulin-dependent signal transduction: effects of PEP-19, neuromodulin, and neurogranin on enzyme activation and cellular homeostasis.

Neuromodulin (GAP-43), neurogranin (RC3), and PEP-19 are small acid-stable proteins that bind calcium-poor calmodulin through a loosely conserved IQ-motif. Even though these proteins have been known for many years, much about their function in cells is not understood. It has recently become appreciated that calmodulin activity in cells is tightly controlled and that pools of otherwise free calmodulin are sequestered so as to restrict its availability for activating calcium/calmodulin-dependent enzymes. Neuromodulin, neurogranin, and PEP-19 appear to be major participants in this type of regulation. One way in which they do this is by providing localized increases in the concentration of calmodulin in cells so that the maximal level of target activation is increased. Additionally, they can function as calmodulin antagonists by directly inhibiting the association of calcium/calmodulin with enzymes and other proteins. Although neuromodulin, neurogranin, and PEP-19 were early representatives of the small IQ-motif-containing protein family, newer examples have come to light that expand the number of cellular systems through which the IQ-peptide/calmodulin interaction could regulate biological processes including gene transcription. It is the purpose of this review to examine the behavior of neuromodulin, neurogranin, and PEP-19 in paradigms that include both in vitro and in situ systems in order to summarize possible biological consequences that are linked to the expression of this type of protein. The use of protein:protein interaction chromatography is also examined in the recovery of a new calmodulin-binding peptide, CAP-19 (ratMBF1). Consistent with earlier predictions, at least one function of small IQ-motif proteins appears to be that they lessen the extent to which calcium-calmodulin-dependent enzymes become or stay activated. It also appears that these polypeptides can function to selectively inhibit activation of intracellular targets by some agonists while simultaneously permitting activation of these same targets by other agonists. Much of the mechanism for how this occurs is unknown, and possible explanations are examined. One of the biological consequences for a cell that expresses a calmodulin-regulatory protein could be an increased resistance to calcium-mediated toxicity. This possibility is examined for cells expressing PEP-19 and both anatomical and cell-biological data is described. The study of IQ-motif-containing small proteins has stimulated considerable thought as to how calcium signaling is refined in neurons. Current evidence suggests that signaling through calmodulin is not a fulminating and homogenous process but a spatially limited and highly regulated one. Data from studies on neuromodulin, neurogranin, and PEP-19 suggest that they play an important role in establishing some of the processes by which this regulation is accomplished.

Calmodulin-binding peptide PEP-19 modulates activation of calmodulin kinase II In situ.

PEP-19 is a 6 kDa polypeptide that is highly expressed in select populations of neurons that sometimes demonstrate resistance to degeneration. These include the granule cells of the hippocampus and the Purkinje cells of the cerebellum. Its only identified activity to date is that of binding apo-calmodulin. As a consequence, it has been demonstrated to act as an inhibitor of calmodulin-dependent neuronal nitric oxide synthase in vitro, although PEP-19 regulation of calmodulin-dependent enzymes has never been characterized in intact cells. The activation of the calmodulin-dependent enzyme calmodulin kinase II (CaM kinase II) was studied in PC12 cells that had been transfected so as to express physiological levels of PEP-19. The expression of PEP-19 yielded a stable phenotype that failed to activate CaM kinase II upon depolarization in high K(+). However, CaM kinase II could be fully activated when calcium influx was achieved with ATP. The effect of PEP-19 on CaM kinase II activation was not attributable to changes in the cellular expression of calmodulin. The cellular permeability of the transfected cells to calcium ions also appeared essentially unchanged. The results of this study demonstrated that PEP-19 can regulate CaM kinase II in situ in a manner that was dependent on the stimulus used to mobilize calcium. The selective nature of the regulation by PEP-19 suggests that its function is not to globally suppress calmodulin activity but rather change the manner in which different stimuli can access this activity.

Identification of a neuronal calmodulin-binding peptide, CAP-19, containing an IQ motif.

Neurons produce polypeptides which can bind the calcium-poor or pre-activated form of calmodulin. It is expected that this class of peptide will serve an important role in maintaining cellular homeostasis since it would modulate calcium-dependent target regulation and redirect intracellular signaling. The lack of conserved sequence has made the identification of these peptides difficult, consequently leading us to exploit their property of binding calcium-poor calmodulin as a means of finding new species. A new peptide termed Calmodulin-Associated Peptide-19 (CAP-19) was purified and characterized. The protein-sequence information was employed in order to recover a cDNA clone from rat which included the entire reading frame for the peptide. Like its counterparts, neuromodulin (GAP-43), neurogranin (RC3) and PEP-19, it contains an IQ motif although the remainder of the peptide is quite different. Northern blot analysis of ribonucleic acid (RNA) from animals of differing ages indicated that the message appears at birth and then persists into adulthood. Antibodies to synthetic peptide were employed for localizing CAP-19. The results indicated that the peptide was localized to neurons in several brain regions. CAP-19 is similar to other calmodulin-binding proteins in that the domain spanning the IQ motif was demonstrated to participate in binding to calmodulin. Database searching showed CAP-19 to be homologous to the silkworm protein, multiprotein bridging factor 1 (MBF1). This homology suggests a potential new role for calmodulin-associated proteins in cellular homeostasis.

Profiling of endogenous peptides as a tool for studying development and neurological disease.

The use of a method to follow changes in endogenous peptide production, as they occur in biological studies, is an excellent complement to other molecular techniques. It has the unique ability to characterize peptides that have been produced from protein precursors, and instrumentation is available that provides high resolution peptide separations that are quantitative, sensitive, and amenable to automation. All tissues express a large number of peptide species that can be visualized, or profiled, on chromatographic separations using reverse-phase high-performance liquid chromatography. This large number of peptides offers many potential molecules that can be used to identify biological mechanisms associated with experimental paradigms. Peptide analysis has been used successfully in many types of studies. In this review, we outline our experience in using peptides as biological markers and provide a description of the evolution of peptide profiling in our laboratories. Peptide expression has been used in studies ranging from how brain regions develop to identifying changes in disease processes including Alzheimer's disease and models of stroke. Some of the findings provided by these studies have been new pathways of peptide processing and the identification of accelerated proteolysis on proteins such as hemoglobin as a function of Alzheimer's disease and brain insult. Peptide profiling has also proven to be an excellent technique for studying many well-known nervous system proteins including calmodulin, PEP-19, myelin basic protein, cytoskeletal proteins, and others. It is the purpose of this review to describe our experience using the technique and to highlight improvements that have added to the power of the approach. Peptide analysis and the expansion in the instrumentation that can detect peptides will no doubt make these types of studies a powerful addition to our molecular armamentarium for conducting biological studies.

Camstatins are peptide antagonists of calmodulin based upon a conserved structural motif in PEP-19, neurogranin, and neuromodulin.

Unbridled increases in intracellular ionized calcium can result in neuronal damage and death. Since many of the deleterious effects of calcium are mediated by calmodulin, we have sought to identify neuronal proteins that inhibit activation of this ubiquitous protein. PEP-19 is a 7.6-kDa neuron-specific protein, which contains a motif similar to the calmodulin binding domains of neuromodulin (GAP-43) and neurogranin (RC3). Here we show that PEP-19 binds calmodulin in an analogous calcium-independent manner with an apparent Kd near 1.2 microM. Furthermore, using the calmodulin-dependent enzyme neuronal nitric oxide synthase, we demonstrate that native PEP-19 is also an antagonist of enzyme activity. Based on the PEP-19 sequence, a series of peptide calmodulin antagonists termed camstatins were synthesized. These analogs define the minimally active domain of PEP-19 and provide a structure/activity relationship for calmodulin antagonism. There was a positive correlation between the binding affinities of the camstatins for calmodulin and their potencies as neuronal nitric oxide synthase inhibitors. Despite the similar IQ motif in PEP-19 and neuromodulin or neurogranin, PEP-19 was not a substrate for protein kinase C. The properties of PEP-19 suggest that it could fulfill a role in neuroprotection.

The dendritic peptide neurogranin can regulate a calmodulin-dependent target.

Neurogranin, a peptide capable of binding the calcium-poor form of calmodulin, was tested in vitro for its ability to modulate a typical calmodulin target. The target employed was the calcium/calmodulin-dependent form of nitric oxide synthase, which is produced by several different types of neurons. Neurogranin for the study was purified from perchloric acid-soluble calf brain proteins by a combination of calmodulin-Sepharose affinity chromatography and reverse-phase HPLC. The protocol yielded highly purified neurogranin that was active in assays using purified nitric oxide synthase. The titration of the enzyme activity with neurogranin demonstrated a concentration-dependent effect of the peptide on enzyme activation. Subsequent analysis of the ability of increased calcium concentrations to activate the enzyme was performed in the presence of different amounts of neurogranin. The effect of neurogranin on the calcium-dependent activation of the enzyme was to depress enzyme activity in the range of 0.2 to approximately 1 microM calcium. Treatment of the neurogranin peptide with protein kinase C eliminated its inhibition on nitric oxide synthase activation. Treatment of the protein kinase C-phosphorylated peptide with calcineurin did not restore the ability of neurogranin to inhibit enzyme activity, whereas treatment with alkaline phosphatase did restore this ability. These results suggest that neurogranin may serve as a member of a unique class of endogenous calmodulin inhibitor that functions to regulate the activation of calmodulin-requiring targets in neurons.

Neuromodulin (GAP-43) can regulate a calmodulin-dependent target in vitro.

The calmodulin-binding polypeptide neuromodulin (GAP-43) was tested in vitro for its ability to modulate a typical calmodulin target, the enzyme nitric oxide synthase. The titration of enzyme with increasing neuromodulin concentrations demonstrated a concentration-dependent decrease in enzyme activity. Subsequent analysis of the ability of increased calcium concentrations to activate the enzyme was tested in the presence or absence of neuromodulin. The effect of neuromodulin on the calcium-dependent activation of the enzyme was to depress enzyme activity in the range of 0.2 to approximately 6 microM calcium. Treatment of the neuromodulin polypeptide with protein kinase C eliminated its ability to inhibit nitric oxide synthase activation. Subsequent treatment of the phosphorylated neuromodulin with calcineurin (phosphatase 2b) caused it to regain its inhibitory action on the enzyme. The results from these in vitro studies have indicated that neuromodulin has the ability to affect the activation of a calmodulin-dependent enzyme at levels of the polypeptide that exist in neurons. They also demonstrated that the regulation occurred within a physiological range of calcium concentrations. Since the inhibition of enzyme activity appeared to be occurring through the interaction of neuromodulin with calmodulin, it seems likely that neuromodulin has a general ability to impede activation of calmodulin-dependent targets.

Increased levels of hemoglobin-derived and other peptides in Alzheimer's disease cerebellum.

Several studies point to the importance of peptides and proteolysis in Alzheimer's disease (AD). Because of its ability to study small proteins and peptides, reverse-phase HPLC was employed to study these species in AD. Cerebellum was chosen for these initial studies because it does not show significant neuronal loss but does show some pathology in AD. Examination of over 600 peptide peaks per case revealed 15 that were elevated in AD. Nine were fragments of hemoglobin, and the remainder included two species of calmodulin, two of myelin basic protein, and one each of 67 kDa neurofilament protein and PEP-19. The cleavage sites on hemoglobin were after hydrophobic residues and immunolocalization was seen preferentially around blood vessel walls and granule cells. The elevation of the non-serum-derived peptides was characteristic of general metabolic changes that occurred in AD cerebellum, and the presence of elevated hemoglobin polypeptides indicated either possible disruption of the blood-brain barrier or selective evasion of it by peptidaceous products. Further studies are required to establish whether hemoglobin fragments have a role in neurodegenerative processes such as AD.

Profiling of endogenous brain peptides and small proteins: methodology, computer-assisted analysis, and application to aging and lesion models.

Significant advances in the technology for the isolation of peptides and small proteins have permitted their identification as biologic markers and enhanced the study of the posttranslational life of proteins. The protocol described here examined large numbers of tissue-derived peptides and small proteins, extracted in low pH and boiled so that proteolysis was interrupted. These were then fractionated batchwise using size exclusion and ion-exchange chromatography. Profiles of species in the peptide pools were then generated on reverse-phase high-performance liquid chromatography (HPLC). The HPLC profiles were evaluated with chromatographic analysis software to identify and quantify peptide peaks and with data compilation programs to sort this information into spreadsheets for comparison of profiles among groups. Using rodent brain, the effects of postmortem delay or age were examined. Postmortem delay produced limited alterations to the profiles, but the effect of age was more pronounced. Many changes were apparent until 12 months, after which the profiles became more constant. Additional peptide profiling of the hippocampus demonstrated changes in peptide content as a function of perforant pathway ablation. The major strengths of HPLC-mediated peptide profiling are that it lends itself to automation and can be used to detect changes in peptides and small proteins among experimental groups or subjects without any prior assumptions concerning which ones might be altered.

Partial sequence of human plasma glutathione peroxidase and immunologic identification of milk glutathione peroxidase as the plasma enzyme.

Plasma glutathione peroxidase (p-GSHPx) is a unique selenoglycoprotein. A hepatic cell line synthesizes both this extracellular form for secretion and the cellular form that remains within the cells. Because the two forms could be a result of post-translational modifications of a product of a single gene, we partially sequenced p-GSHPx. Purified p-GSHPx was trypsin digested, and three of the peptides were sequenced. Only one of the peptide sequences was partially homologous to a sequence found in human cellular glutathione peroxidase. Because p-GSHPx is a secreted enzyme, we determined whether GSHPx in milk (another extracellular fluid) is due to this form of the enzyme. Ninety percent of human milk GSHPx activity could be precipitated by anti-p-GSHPx-immunoglobulin G. Thus, most, if not all, GSHPx activity in milk is due to the plasma selenoprotein form of the enzyme. In milk of two North American women, 3.6% and 14.3% of selenium was associated with GSHPx.

The amino terminus of the putative Drosophila choline acetyltransferase precursor is cleaved to yield the 67 kDa enzyme.

A putative precursor of the 67 kDa choline acetyltransferase (Acetyl-CoA: choline-O-acetyltransferase; EC 2.3.1.6) polypeptide from Drosophila was examined using polyclonal antibodies. The central purpose of the study was to probe the suspected precursor with anti-peptide antibodies that could identify a cleavable amino terminal domain, since such a structure could be responsible for targeting the enzyme to the presynaptic terminal. Antisera were produced to both a plasmid-expressed fusion-free enzyme protein and a 26-amino acid-long peptide reproducing sequence from the enzyme. Both antisera were capable of precipitating enzyme activity from crude supernatants. Western blotting with the antibody to the plasmid-expressed enzyme visualized a major polypeptide at 75 kDa and minor polypeptides at 67 and 54 kDa. Affinity-purified IgG to the synthetic peptide only recognized the 75 kDa component and was unable to recognize purified 67 kDa enzyme protein. Timed autolysis of the enzyme in crude homogenates demonstrated both a 67 kDa polypeptide that was present prior to homogenization and a species that appeared as a product of the autolysis. The evidence from this study is consistent with the expectation that the 75 kDa band, visualized on Western blots with antisera to the enzyme, is an authentic enzyme protein. These data further suggested that the 75 kDa protein is an amino-terminally extended precursor of the 67 kDa enzyme that can be cleaved to generate the 67 kDa species.

Sequence analysis of a proteolyzed site in Drosophila choline acetyltransferase.

The amino terminal sequence of the 13-kilodalton (kD) polypeptide present in purified Drosophila acetyl-CoA: choline-O-acetyltransferase (EC 2.3.1.6) was determined, and its position in the sequence of the intact enzyme was located. Enzyme polypeptides for sequencing were obtained from native enzyme protein by denaturation, followed by fractionation on reverse-phase HPLC. The 13-, 54-, and 67-kD polypeptides recovered from the separation were subjected to amino terminal sequencing. Only the 13-kD fragment yielded a sequence. The 67- and 54-kD polypeptides appeared completely blocked to gas-phase Edman sequencing. The location of the amino terminal sequence from the 13-kD polypeptide in the cDNA-deduced enzyme sequence indicated that this fragment represents the carboxyl portion of the 67-kD enzyme, with the 54-kD polypeptide providing the amino terminal portion. The proteolysis that gave rise to the 13-kD polypeptide occurred at the carboxyl side of a monobasic lysine residue. An earlier comparison of the enzyme from Drosophila and pigs indicated that the cleaved lysine may be a conserved residue in the porcine enzyme. The cleaved enzyme region characterized in this study does not coincide with the regions of high homology found in the two enzymes, but hydrophilicity profiles generated for this area showed similarities.

Evidence for the transneuronal regulation of cerebellin biosynthesis in developing Purkinje cells.

We have investigated the expression of a unique class of neuropeptides, the cerebellins, in normal and neurodevelopmentally mutant mice. Employing HPLC separation, gas-phase Edman sequencing, and immunocytochemistry, the normal Balb/c mouse cerebellum is shown to contain 2 Purkinje cell-specific neuropeptides, cerebellin and des-Ser1-cerebellin. In this strain of mouse the cerebellins appear during early postnatal development and their subsequent levels parallel the most dramatic period of cerebellar development: granule cell migration and parallel fiber formation, synaptogenesis, Purkinje cell dendritic maturation, and establishment of adult cytoarchitecture. In mutant mice (reeler, weaver, and staggerer), in which these early developmental events are markedly disrupted, Purkinje cells contain much lower levels of cerebellin and des-Ser1-cerebellin. In general there is a correlation between the formation and number of parallel fiber-Purkinje cell synapses and cerebellin levels. For example, the staggerer mutant, which totally lacks these synapses, is essentially devoid of cerebellin, whereas in reeler, cerebellin seems to be dependent upon the position of individual Purkinje cells and their ability to form contacts with granule cells. These results indicate that granule cells can modulate the level of cerebellin in Purkinje cells. A number of models to explain these data are discussed.

Cerebellin and related postsynaptic peptides in the brain of normal and neurodevelopmentally mutant vertebrates.

The rat cerebellum was previously shown to contain two polypeptides, a hexadecapeptide termed cerebellin and an apparent metabolite des-Ser1-cerebellin. The cerebellins have a high degree of sequence homology with residues 625-641 of the polyimmunoglobulin (polyIg)-receptor adjacent to its membrane-spanning domain. Since the cerebellins are localized in Purkinje cells and enriched in synaptosomes, this might indicate that cerebellin is a specific proteolytic cleavage fragment of a synaptic protein involved in the transcytosis of an unknown ligand. Using a specific cerebellin radioimmunoassay described here combined with high-performance liquid chromatography, cerebellin immunoreactivity could be demonstrated in the cerebella of all vertebrates examined from man to chicken. Cerebellin immunoreactivity is localized to Purkinje cells in the rat, mouse, and chicken. Furthermore, cerebellin expression is under developmental regulation in both the chicken and mouse. In addition, neurodevelopmental mutations of mice that eliminate granule cells cause a large deficit in cerebellin levels, suggesting some form of transneuronal regulation.

Production of polyclonal antisera to choline acetyltransferase using a fusion protein produced by a cDNA clone.

A fusion protein containing a Drosophila choline acetyltransferase (ChAT) cDNA insert was purified from a lambda gtll lysate of Escherichia coli. The cDNA insert, which contained a 728-amino acid coding region for ChAT, was used for immunizing rabbits. Three different antisera were produced that could recognize native Drosophila ChAT with low titer. In addition, all three antisera stained enzyme polypeptides using the Western blot technique at high titers. The antisera recognized ChAT polypeptides with molecular masses of 67 and 54 kilodaltons in Western blots of partially purified enzyme; these polypeptides had previously been identified using monoclonal anti-ChAT antibodies and are the major components of completely purified enzyme. It was surprising that when these antisera were used to stain Western blots of Drosophila head homogenates, the major immunoreactive band had a molecular mass of 75 kilodaltons. The relationship of this 75-kilodalton polypeptide to ChAT activity was investigated by fractionating fresh fly head homogenates using rapid HPLC gel filtration chromatography. Analysis of column fractions for enzyme activity and immunoreactive polypeptides indicated that the 75- and 67-kilodalton polypeptides can be resolved and are both enzymatically active. In addition, a correlation was observed between the relative immunostaining intensities of both the 75- and 67-kilodalton bands and ChAT activity when supernatants from fresh fly head homogenates were autolyzed at 37 degrees C. Our results indicate that ChAT is present in fresh Drosophila heads primarily as an active enzyme with a molecular mass of 75 kilodaltons.(ABSTRACT TRUNCATED AT 250 WORDS)