A protein/peptide chemistry core facility
|Director:||David B. Teplow, Ph.D.|
|Staff:||Margaret M. Condron|
|Location:||Neuroscience Research Building|
635 Charles E. Young Dr. South (Room 455)
Los Angeles, CA 90095
Mail Code 172216
General Information: The Biopolymer Laboratory is a core facility that provides peptide and protein chemistry services to the UCLA community and to other researchers (nationally and internationally). These services include quantitative amino acid analysis, protein sequencing, peptide synthesis, and mass spectrometry.
Amino Acid Analysis (AAA): Determines the amino acid composition of proteins and peptides. AAA is the most accurate method for protein quantitation.
Method: Amino acid compositions generally are determined by initial hydrolysis of samples using vapor-phase HCl. The free amino acids then are fractionated and quantitated by reverse phase (RP)-HPLC following pre-column derivatization with a highly fluorescent amino-reactive probe. System sensitivity is approximately 1 pmole. AAA reports provide the amounts of each amino acid in grams and moles.
Sample preparation: Samples may be provided as liquids or solids or immobilized on chemically-inert solid supports (e.g., polyvinylidene difluoride membranes (PVDF)). Volatile aqueous or organic solvents are preferred. Although not necessary, prior knowledge of the sequence or composition of a sample can be useful for optimizing hydrolysis or analysis conditions. The amount of sample required varies inversely with the molecular weight of the sample.
Cost: $65 per sample, which includes sample preparation, hydrolysis, and analysis. For greatest accuracy, we recommend analysis of triplicate samples.
Protein Sequence Analysis (PSA): Determines the sequence of amino acids beginning from the N-terminus of a sample. Analyses can be done using unlabeled or radiolabeled material.
Method: Automated protein sequencing is done using a state-of-the-art pulsed-liquid solid phase sequenator with on-line phenylthiohydantoin (PTH)-amino acid analysis (Applied Biosystems cLC system). System sensitivity is approximately 1 picomole. Radiosequencing also can be done. In this case, substantially lower amounts of protein are required. Labels may be introduced into the protein cotranslationally, through the use of 3H-, 14C-, 35S-labeled amino acids, or postranslationally through chemical or enzymatic modification (e.g., phosphorylation). Other labels may also be utilized, as appropriate.
Sample preparation: Samples may be provided as liquids or solids or immobilized on chemically-inert solid supports (e.g., polyvinylidene difluoride membranes (PVDF)). Ideally, 10- 100 picomoles of material should be provided. However, required amounts vary both with protein molecular weight and amino acid sequence. Small amounts of highly pure, low molecular weight peptides can be sequences at the sub-picomole level. In contrast, proteins exceeding 50-100 kDa may require isproportionately large amounts should lengthy sequence information be required.
Cost: A $250 minimum fee supports sample preparation, instrument set-up, and the initial 5 cycles of sequencing. Each cycle thereafter costs $40. The initial five cycles of Edman chemistry provide information on whether, and at what levels, sequence information is being produced. In the event no sequence information is obtained, the background levels of PTH- amino acid derivatives produced during this short initial run provide useful information about whether the sample was blocked or simply was present in insufficient amount.
Solid Phase Peptide Synthesis (SPPS): Chemical synthesis of peptides provides the means to tailor-make natural, unnatural, and isotope-labeled peptides.
Method: Peptides are synthesized according to the general strategy of Merrifield et al. using automated solid-phase FMOC chemistry. Peptides routinely are produced that range in length from 2-50 amino acids. Control of peptide synthesis quality is accomplished through a combination of AAA and RP-HPLC. PSA or liquid chromatography-mass spectrometry (LC-MS) characterization are also available. Peptides are provided as lyophilizates ready for solubilization. Standard syntheses are done at a 100 micromole scale.
Custom Synthesis Options: A variety of custom synthesis options are available, including N- terminal (acylation) or C-terminal (amidation) modification, incorporation of unnatural amino acids (depending on availability), and use of stable isotope-labeled amino acids for preparation of NMR analytes.
Cost: Standard syntheses use single coupling chemistry and cost $300 for instrument set up and $60 per residue. Additional costs accrue for custom synthesis options or for modifications to the chemistry necessary to produce peptides containing “difficult sequences.” Synthesis design is done pre facto with the user to ensure successful and economical peptide production.
Policy: The Biopolymer Laboratory (BPL) will synthesize peptides corresponding to the sequence and end-groups requested by the user. Cost estimates for syntheses are based on peptide work-up expenses and the number of amino acids in the peptide. The BPL may alter the synthesis chemistry in order to produce superior products, in which case additional costs may accrue.
The BPL will perform quality control (QC) analyses on the end product to show that the correct molecules have been made. The QC procedures include amino acid analysis, reverse phase HPLC, and LC/MS.
The BPL will synthesize essentially any sequence requested, but cannot be responsible for the biophysical behavior of the resulting molecules. These behaviors may include, but are not limited to:
- Lack of biological activity
- Aggregation, especially in the presence of cysteine residues
The BPL requires that users understand and agree to these policies prior to the performance of any peptide syntheses.
Mass Spectrometry (MS): Determines the molecular weight or sequence of proteins and peptides.
Method: Samples are analyzed using a ThermoFinnigan LCQDECA mass spectrometer. Analytes are introduced into the mass spectrometry either by direct infusion or RP-HPLC. For RP-HPLC, peptides and peptide digests typically are chromatographed using gradients of acetonitrile in 0.1% (v/v) glacial acetic acid and 0.02% (v/v) trifluoroacetic acid in water at a flow rate of 100 µl/min on 150 mm x 1.0 mm, 5 µm particle size, C18 or diphenyl columns. Elution is monitored by multi-channel UV absorbance at 214, 225 and 254 nm. Other wavelengths can be used, if appropriate. Mass spectra are acquired in positive ion mode using a mass range (m/z) of 200–2000.
Sample preparation: Samples may be provided either in solution or as solids. Solutions should be composed of volatile components.
Cost: Infusion—$100 per sample. RP-HPLC-MS—$200 per sample.
Ordering/Additional Information: Please contact Margaret Condron by email (firstname.lastname@example.org). Our response time generally is <24 hours, not including weekends and UCLA holidays. Samples may be dropped off at the Biopolymer Laboratory from 10am–3pm, Monday- Friday, or at other times by prior arrangement. Please note that a 25% surcharge is required of non-academic users.