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Core Director: Joseph R. Reeve, Jr., Ph.D., Professor,
Division of Digestive Diseases, UCLA
Core Co-Director: David
Keire, Ph.D., Visiting Associate Researcher, UCLA Department of
Medicine, Gordon Ohning, M.D., Ph.D.
OBJECTIVES
The three main objectives of the
Peptidomics Core are to (1) train CURE investigators in the use
of peptides and molecular biological techniques for their own investigations,
(2) provide instrumentation and/or services that are too costly
or too time consuming to be performed in individual laboratories,
and (3) provide products necessary for CURE investigators to conduct
their own research.
CORE SERVICES
A) CHARACTERIZATION OF NATURAL OR SYNTHETIC PEPTIDES
A1) TRAIN CURE: DDRCC INVESTIGATORS IN THE PROPER HANDLING OF PEPTIDES
Every peptide used by CURE: DDRCC investigators (cholecystokinin, gastrin releasing peptide, PACAP, CRF, etc.) is a unique reagent that requires proper handling to obtain valid results free from artifacts. Investigators are taught which buffers are suitable for stock solutions of peptides, the best buffers for diluting stock solutions, and the methods to demonstrate peptide amounts and stability in these solutions (HPLC, HPCE, amino acid analysis and absorbance at 280 nm). Investigators are advised about how to deliver peptides into their experiments and how to evaluate losses or modifications during delivery. All CURE: DDRCC investigators can consult with the Core directors or staff about proper utilization of peptides. This is by far the most utilized service of the Peptidomics and Proteomics Core.
A2) CHARACTERIZE SYNTHETIC PEPTIDES PRODUCED AT CURE: DDRCC OR AT OUTSIDE VENDORS BY HIGH PERFORMANCE CAPILLARY ELECTROPHORESIS
High performance capillary electrophoresis (HPCE) is an analytical technique for analysis of peptides and other biologically active molecules. It is especially suited for detection of contaminants of synthetic or natural peptides purified by reverse phase HPLC. This is due to the fact that it is an entirely different separation mode from reverse phase chromatography and it requires extremely small samples. Less than 10 microliters are used for the sample and only a few nanoliters are applied to the column. The importance and value of using dual approaches to peptide characterization has been established; the largest data base for detecting impurities is with peptides corresponding to portions of HIV proteins. About 70% of the proteins had similar levels of impurity detected by HPLC and HPCE. About 20% of the peptides had impurities between 10-20 percent, and 10% of the peptides had impurities between 30-70 percent. While HPCE is available for training CURE: DDRCC investigators, this service is provided free to investigators who only require a few routine samples.
B) MICROSEQUENCE ANALYSIS OF NATURAL PEPTIDES AND PROTEINS
The PRP Core does not operate a microsequence analysis instrument, but Dr. Reeve has collaborated for more than 20 years with John E. Shively, a consulting CURE: DDRCC member, on microsequence analysis at the City of Hope Research Institute, Duarte, California.(3) Peptide microsequence analysis is provided on a charge-back basis paid directly to the facility performing the service. Peptides purified from reverse phase HPLC are evaluated by HPCE to insure they are suitable for microsequence analysis. Purified fractions that contain primarily a single peptide and are sufficiently concentrated (50 pmol/5l) are loaded directly onto a microsequencer. Peptides that are too dilute are concentrated on a microbore HPLC before they are loaded on the microsequencer. The Core Director assists CURE: DDRCC investigators in purifying samples until they are suitable for microsequence analysis, assures that the samples are properly delivered, and helps investigators interpret results.
Several CURE: DDRCC investigators' projects require microsequence analysis of proteins after purification by 2D gel electrophoresis; examples include signal transduction proteins, enzymes and receptors or fragment peptides from these proteins. Microsequence is especially valuable in determining modification sites when peptides have multiple consensus sites for the modification. Proteins can be sequenced from gels after washing away the dye by rinsing with digestion buffer. The protein is then digested with trypsin and the products are either analyzed by on-line LC/MS/MS4 or purified by HPLC and fractions characterized by microsequence analysis. The minimum amounts for this type of protein analysis are 100 picomole, which can easily be recovered from a single 2D gel. Our collaborators at the City of Hope have determined the entire sequence of some proteins using this type of technique with multiple specific endopeptidases. However, for most analyses it will be quicker and more cost effective to make molecular probes to sequences determined by analysis of tryptic peptides and determine the entire sequence through molecular biological techniques. The Director and Co-Director of the Core will assist investigators in deciding which approach to use in determining the sequence of new proteins.
C) PURIFICATION OF COMPLEX MIXTURES OF PROTEINS AND PEPTIDES
Over the years the most productive efforts of the Core have been the purification and characterization of natural peptides from stomach, intestine, brain, and tumors from various species (4). These characterizations have been carried out in collaboration with many investigators. The purifications have provided the basis for training several postdoctoral fellows and young investigators in analytical and preparative chromatography by reverse phase, gel permeation, or ion-exchange with high or low pressure systems. The PRP Core has liquid chromatography techniques complementing 2D gel purification of proteins. These techniques allow 3D-4D separations by using size exclusion, cationic, anionic, and reverse phase (with several gel matrices with many buffers) separations (5;6). Natural peptide and protein purification is performed on a collaborative basis.
D) CUSTOM PEPTIDE SYNTHESIS AND SYNTHESIS OF G-PROTEIN SEGMENTS COUPLED TO PEPTIDE CARRIERS (INHIBITORS OF G PROTEINS)
The principal objectives of peptide synthesis are to provide CURE: DDRCC investigators with 1) synthetic peptides, 2) design strategies for distinctive peptide reagents, and 3) state-of-the-art purification and analysis of synthetic peptides. Specialized services include synthesis of unusually long peptides (longer than 25 residues), synthesis of peptides with unique amino acid analogs, and the availability of a professional synthetic peptide chemist to help design peptides that are best suited for specific research needs. The director of the Core assists investigators in designing peptides that meet the requirements of their particular study and advising them on whether their peptide utilization is more suited for large or small-scale synthesis. The Core operates a state-of-the-art peptide synthesizer that monitors deprotection steps (ABI 433A). This instrument is especially suitable for large amounts of peptide or for long peptides that are difficult to synthesize. Peptides included in this category are exemplified by canine CCK-58 (7). Peptides produced by the CORE will be done on a collaborative basis, or as a service without collaboration. The Core will assist CURE: DDRCC investigators in need of small amounts of short peptides with ordering these peptides with outside vendors and will analyze peptides obtained from these vendors Synthetic peptides corresponding to the third helix of the antennapedia homeodomain can internalize attached reagents through receptor independent mechanisms (8). We have synthesized the 16 amino acid peptide coupled to portions of intracellular reagents. In collaboration with Drs. Rey and Rozengurt, we have preliminary data indicating that portions of PKC can be synthesized with the carrier peptide and when incubated with cells block the ability of intracellular PKC to phosphorylate and activate protein kinase D (PKD). These technologies will be developed in collaboration with the Signal Transduction Core.
E) CIRCULAR DICHROISM AND NUCLEAR MAGNETIC RESONANCE OF PEPTIDES
E1) CIRCULAR DICHROISM OF PEPTIDES
Circular dichroism (CD) determines the secondary structure content of peptides and proteins in solution by measuring differential absorption of plane polarized light (ellipticity). CD is useful in determining if an amino acid substitution or modification changes the overall conformation of a protein. This has been used to show that the removal of the amino terminal two amino acids from PYY causes profound changes in conformamtion (9). The relative amounts of helical, β-sheet and random coil conformations can be calculated from characteristic ellipticities. For example, a helical peptide has bands at [+]195, [-]208 and [-]222 nm and the 222 nm band can be used to calculate the percent helicity. Typical sample concentrations range from 0.1 to 1 mM in a 200 microliter (0.1 mm pathlength) cell.
E2) NUCLEAR MAGNETIC RESONANCE (NMR) OF PEPTIDES
NMR methods are used to determine the primary, secondary and tertiary structure of peptides and proteins in solution (10). One and two-dimensional (1D and 2D) NMR experiments are used to assign the chemical shifts of all peptide protons. Nuclear Overhauser effect (NOE) and spin-spin scalar coupling interactions between proton pairs are measured and used to determine distances and angles, respectively. The set of distance and angular constraints are input into molecular modeling programs to calculate a tertiary structure if a unique fold exists. Typical sample concentrations for structure determination are 1 to 2 mM in a 750 microliter 5 mm sample tube for the 500 MHz instrument.
F) RADIOIMMUNOASSAY PROCEDURES
F1) RADIOLABELING OF PEPTIDES FOR RADIOIMMUNOASSAY AND RADIO-RECEPTOR STUDIES
Two types of peptide radiolabeling are available for CURE: DDRCC investigators: (1) binding of radioactive Bolton-Hunter reagent to primary amino groups, and (2) iodination of peptides containing tyrosine or sometimes histidine. The Core staff has extensive experience in producing labeled peptides capable of binding receptors even in difficult cases such as those with an oxidizable methionine in the active region of the peptide (Bolton-Hunter labeling is the best technique in these cases). Peptides with several amino groups, some in the region of receptor binding, needed for receptor studies can have their groups oxidized during iodination reduced (11). The Core provides labeled peptides for the cost of reagents (no labor charges) to investigators who need them occasionally. Investigators with frequent needs for labeled peptides are trained by the Core staff in methods for labeling and purification.
F2) TRAINING IN ELISA, WESTERN BLOTTING, AND ANTIBODY PURIFICATION
The Core provides teaching services for techniques and procedures including Western blot analysis, radio-iodination, ELISA, design of antigens, antibody purification, and RIA procedures. These services are used by CURE: DDRCC investigators, technicians, and postdoctoral fellows.
F3) RADIOIMMUNOASSAY OF PEPTIDE SATIETY FACTORS (PYY, CCK AND GHRELIN) AND PROVIDE EXPERTISE AND EQUIPMENT FOR OTHER RADIOIMMUNOASSAYS
It is our experience that most requests for radioimmunoassays come for the same assays from the same investigators. Requests are usually for very large numbers of samples for funded projects. One special set of peptides are those that regulate feeding. The Core will provide radioimmunoassay for cholecystokinin, PYY(3-36) (this will require chromatography as well as RIA) and ghrelin. A new method of processing blood that provides quantitative extraction of all molecular forms of circulating peptides has been recently developed with cholecystokinin (12); the method allows new insight into the molecular forms and concentrations of endocrine cholecystokinin . This method will be used to evaluate PYY(3-36) and ghrelin blood concentrations. Because of budget limitations the Core will perform other assays requested only if the numbers of samples are limited. For infrequent users with large numbers of samples the Core will provide the radiolabel that has been purified by HPLC and evaluated by binding checks and standard curves. For frequent users the Core will provide the equipment necessary for iodination of peptide tracers including iodination hood, G-10 sizing columns and HPLC columns and equipment for purification of labels. The Core maintains the necessary equipment for radioimmunoassays, including dilutors, centrifuges, and gamma counters, and schedules use of this equipment. The Core Director and staff are available to train novices, to trouble shoot problems in the assay, and to assist all CURE: DDRCC investigators.
F4) DISTRIBUTION OF EXISTING MONOCLONAL AND POLYCLONAL ANTIBODIES
Antibodies prepared by the former Antibody Core will be made available to CURE: DDRCC members and to other investigators outside the Center. These antibodies most often are used for radioimmunoassay or for immunohistochemistry, but certain antibodies are made available in large amounts for immunoneutralization. The cost of obtaining enough antibody for immunization experiments would prohibit many experiments that are now possible with previously produced antibodies. Many antibodies that have been well characterized and used extensively are listed on the CURE: DDRCC web page and are generally available. Peptides that were used for conjugation to produce epitope-specific antisera also are provided to investigators performing Western blotting or immunohistochemistry for specific immunohistochemical absorption. Some specific examples are listed below:
Polyclonal Antibodies Available:
Anti-idiotypic gastrin mouse antibody
Albumin: human and rat bombesin/GRP
EGF: human, mouse, and rat
Enteroglucagon
Endothelin
Enkephalin
Fusion Protein
Gastrin, C and N-terminal
GIP
GRP: gly-extended
Granuliberin
Green fluorescence protein
Insulin-like growth factor
Kinetensin
LAP
LAP: microsomal
Leptin
Levitide
Motilin
Neuromedin B
Neuromedin N
Neurotensin: C and N-terminal
Neutral-endopeptidase
NPY
Pancreastatin, human
Pancreatic polypeptide
Progastrin
RanatensinSecretin
Somatostatin
Substance K
Substance P
Thermolysin
TRHVIP: C and N-terminal
Monoclonal Antibodies Available:
Gastrin/CCK
Gastrin specific
Rat CGRP
Somatostatin
Rat PP
Anti-KLH (control)
VIP
Glucagon
IGF1
Urease
Dog epi-cell-surface
Rat R-1 fibroblast surface
Rat histidine decarboxylase
F5) ASSIST INVESTIGATORS IN OBTAINING CUSTOM ANTIBODIES FROM COMMERICAL VENDORS.
Since the submission of the last CURE: DDRCC proposal, three situations have caused us to abandon the production of our own monoclonal and polyclonal antibodies. 1) The availability of quality antibodies from vendors at low cost. 2) The death of the Antibody Core Director, Dr. John Walsh, who provided the broad experience and expertise in methods required for successful generation of antibodies. 3) The retirement of the the Core Co-Director and major technologist, Helen Wong. The PRP Core will help CURE: DDRCC investigators design antigens for antibody production, contact commercial vendors, and evaluate the antibodies obtained from vendors.
G) INTACT PROTEIN MOLECULAR WEIGHT MEASUREMENTS.
Complete characterization of a protein requires an intact molecular weight measurement so that the presence of any covalent modifications and discrepancies with the calculated mass are recognized (13-15). A number of mass spectrometers and modes of operation are available to determine intact masses of proteins. The type of analysis to be performed will be determined by personnel in the Proteomics Facility after discussion with the CURE investigator requesting molecular weight determination.
MALDI-TOF gives accuracies to  0.1% (up to 200 kD +) MALDI-FTMS gives accuracies to  0.001 % (up to 5 kD) ESI-Quadrupole gives accuracies to  0.01 % (up to 150 kD) ESI-FTMS gives accuracies to  0.001 % (up to 50 kD) LCMS will also be used when required, particularly for mixtures of proteins, when de-salting is required and for membrane proteins for which the facility has particular expertise (see Julian Whitelegge's biosketch).
H) PROTEIN IDENTIFICATION BY MASS SPECTROMETRY
H1) One dimensional and two dimensional gel electrophoresis of proteins: The most commonly used method for displaying complex protein samples is two dimensional polyacrylamide gel electrophoresis with isoelectric focusing in the first dimension and size exclusion in the second dimension, followed by protein staining. Protein identifications can be made on spots excised from such gels (16). Apparatus is available to run one and two dimensional electrophoresis of proteins. Core personnel will either train CURE: DDRCC investigators to use this equipment or run the experiments, depending upon the number of gels that will be required.
H2) Automated in-gel and solution phase reduction/alkylation and enzymatic digestion followed by MALDI-TOF peptide mapping and on-line access to ProteinProspector for mass-tag protein identification via any of the web-based data bases: Large scale proteomics requires that the procedures be automated so that the process is sufficiently fast to handle the work load. The UCLA proteomics lab can be accessed at: www.proteomics.crump.ucla.edu.
H3) Automated in-gel and solution phase reduction/alkylation and enzymatic digestion followed by LC-ESI/MS and MS/MS for sequence-tag protein identification via the SEQUEST/TurboSEQUEST/ Sonar process on the Finnigan LCQDECA or via the MASCOT /Pro ID/ PepSee programs using data collected on the ABI QSTARXL: When the mass tag protein identification approach made with MALDI-TOF data is unsuccessful, or if confirmatory data are required, the sequence tag experiment is performed involving combined LC-MS and LC-MS/MS (17).
I) DETECTION AND IDENTIFICATION OF PROTEIN POST-TRANSLATIONAL MODIFICATIONS (PTMS).
Post-translational modications are important events that influence protein function, and these need to be defined for each protein studied (18;19). For example, reversible protein phosphorylation has emerged as a fundamental mechanism in the signal transduction mechanisms that mediate the biological effect of hormones, neurotransmitters, growth factors and cytokines. Comparison of measured (1 above) and calculated molecular weights will be used to detect the presence and mass of PTMs. Endopeptidase (eg trypsin, Lys C, etc) and CNBr maps of proteins will be used to locate the fragment containing the PTM. MS/MS will be used to identify the residue harboring the PTM.
I1). PROTEIN TRUNCATION
This is of particular interest to CURE: DDRCC investigators. Evidence of truncation will quickly emerge from the comparison between measured and expected molecular weights (1 above), and CNBr mapping will be the first step to locate the mass of the shortened fragment. Further digestion with trypsin and MS/MS may be necessary for unambiguous determination of the truncation site.
I2). PROTEIN PHOSPHORYLATION
This PTM, which is also of particular interest to CURE: DDRCC investigators, results in a mass increment of 80 Da per phosphate group incorporated, and this mass change is readily detected by intact molecular weight measurements (1 above). Localization of the position of the phosphate moiety within the amino acid chain will be achieved by MS/MS following fragmentation of the protein (Gibson, 1990). There is now a burgeoning literature on the application of these techniques to protein phosphorylation studies. When necessary, de-phosphorylation with phosphatases will be used to verify the PTM. Digestion with site-specific reagents (trypsin and CNBr) followed by MALDI-TOF and/or LC-ESIMS will be used to identify those protein fragments which contain the phosphate groups. Post-source decay experiments (PSD) on the MALDI-TOF instrument, or, more likely, MS/MS using collisionally activated dissociation (CAD) on pre-selected parent ions during LC/MS will be used to locate the position(s) of phosphorylation within the relevant tryptic fragments. Interpretation of the MS/MS spectra to localize the position of phosphorylation will be aided by the availability of the MS/MS spectra of the corresponding non-phosphorylated fragments. All available mass spectrometry-based methods will be exploited to sort out the phosphorylation of proteins of interest. Thus, note is taken of the recent improvements in phosphopeptide recovery during immobilized metal affinity chromatography (IMAC) by methyl ester formation, recently reported by Barrett-Wilt et al (2002). This development, which should at least partially overcome the poor recovery of low abundance phosphopeptides by the IMAC procedure observed by us (R.H. Lee, personal communication) and others, makes this a potentially useful approach to pre-concentrate phosphorylated protein fragments prior to PSD or LC/MS/MS. Also, precursor ion scanning will be used when appropriate to identify phosphopeptides as they elute during LCMS. Recognition of the presence of specific protein structural features can be assisted by this scan option that is available on triple quad and QSTAR instruments. The experiment relies on the detection of modification-specific fragment ions produced during CAD of peptides. Specifically, the negatively charged fragment ions at m/z 63 and 79 signify the presence of and moieties characteristic of phosphorylation. Phosphotyrosine can be specifically detected by its corresponding immonium ion at m/z 216.04, Da. but only by instruments such as the QSTAR which have the resolution necessary to distinguish this ion from other extraneous signals around 216 Da that arise from certain dipeptides (Akilish Pandey, personal communication). When ultra-high resolution and mass accuracy is required to resolve ambiguities, we will resort to spectra collected on the FTMS instrument which is equipped with both MALDI and ESI sources. These technologies will interface with those provided through the Signal Transduction Core (e.g. antibodies that detect the phosphorylated state of a specific residue in a given protein) and the GI Cell Biology and Functional Genomics Core. Collectively, they will provide DDRCC members with novel powerful approaches to define PTMs that regulate the function of critical proteins in gastrointestinal cells.
J) RELATIVE PROTEIN ABUNDANCE MEASUREMENTS.
Relative abundance is the most convenient method for incorporating measurements in changes in expression/degradation into the proteomics protocol (20). These will be made using commercially available reagents (e.g., ICAT from ABI which targets free thiol groups) and in-house prepared reagents (e.g., N-acetoxysuccinimide which targets free primary amino groups).
K) MOLECULAR WEIGHT DETERMINATION OF PEPTIDES AND OTHER BIOLOGICAL MOLECULES
Molecular weight measurements by mass spectrometry are one of the standard tools for compound characterization (21;22). Low resolution and high resolution mass spectrometry analysis with EI, CI, FAB, APCI, ESI, and MALDI will be used to determine the molecular weight of peptides and other biological molecules of significance (lipids, sugars, nucleic acids, metabolites etc).
L) MIXTURE ANALYSIS AND COMPONENT QUANTITATION WITH INTERNAL STANDARDS.
Isotope dilution measurements by GC/MS and LC/MS are standard techniques for compound quantification in complex extracts (23). The Core will provide GC-MS (EI, CI) for mixture analysis and component quantitation with internal standards.
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