Protein Science
Biomolecular Research Facility
The Biomolecular Research Facility offers a range of analytical services listed below to study proteins and peptides.
Services:
Protein sequencing
The BRF can perform sequence analysis by mass spectrometry and Edman chemistry. For identification of proteins and obtaining sequence data for probe design, mass spectrometry sequencing of a protein from a gel is the more efficient procedure because of the sensitivity,analysis time and relatively simple sampe preparation. For some analyses, namely determination of the N-terminal, identifiying a proteolysis site, sequencing of proteins blotted to PVDF and analysis of radioactive samples, Edman sequencing is the preferred technique. For further information, contact Dr. Nicholas Sherman 924-0070 or Dr. John Shannon.
Protein sequencing by mass spectrometry
Mass spectrometry can give sequence data from any Coomassie stained band or gel spot. The protein is digested with trypsin in the gel, peptides eluted and fractionated by reverse phase chromatography and introduced into the mass spectrometer. The mass spectrometer determines the mass of the peptides and the sequence (by collisionally induced dissociation). From the masses of the peptide fragments, sequence data is determined by comparison with known sequences or by manual interpretation.
Identifying and sequencing proteins in gels has been very successful here. Electrophoresis eliminates impurities and shows the purity of the protein; immobilization of the protein in the gel allows reduction, alkylation and washing of the protein. Tryptic digestion is the standard procedure for producing peptides because of the reliability of the digestion and production of suitable sized peptides which behave well in a mass spectrometer. Using autolysis resistant trypsin gives only a few well known autolysis products.
Coomassie stained gel bands are the standard samples. We recommend regular Coomassie stain with fixation of the protein. Silver stained gels can be analyzed, if the appropriate staining procedure is used, (see the protocols suitable for sequencing) but contamination, especially with keratin, is often a problem. When submitting a sample, please include a sample form . We prefer to have the whole gel and a picture showing the bands or spots to be analyzed. If shipping the gel from outside the University of Virginia, we suggest sealing it in a bag placed between sheets of cardboard for protection and shipped to:
Dr. Nicholas Sherman
Room 1101 Jordan Hall,
Department of Microbiology, Box 800734,
University of Virginia Health System,
1300 Jefferson Park Avenue
Charlottesville, VA 22908-0734
(phone 434-924-0070)
Our current instruments are a Finnigan LCQ Deca XP and LCQ Classic with micro-spra
y source.
Papers describing the use of the techniques are:
Mandal, A., Naaby-Hansen, S., Wolkowicz, M.J., Klotz, K., Shetty, J., Retief, J.D., Coonrod, S.A., Kinter, M., Sherman, N., Cesar, F., Flickinger, C.J. and Herr, J.C. (1999) Biol. Reprod. 61 1184-1197
S.L. Hanna, N.E. Sherman, M.T. Kinter, J.B. Goldberg (2000) Microbiology 146 2495-2508
A recommended reference on the procedures is:
Protein Sequencing and Identification Using Tandem Mass Spectrometry M. Kinter and N.E. Sherman (Wiley Interscience, 2000)
Protein sequencing by Edman sequencing
N-terminal sequencing, also known as Edman sequencing, is performed on an Applied Biosystems Procise protein sequencer. In this technique, the N-terminal amino acid is derivatized with PITC, cleaved by acid and identified by reverse phase chromatography. Repeating this process gives a protein sequence.
Samples can be supplied in solution or as blots. Samples in aqueous solution usually contain buffers and salts which interfere with the sequencing chemistry, but it is simple to adsorb the protein on to PVDF membrane while the buffers pass through and are washed away. A popular way of preparing proteins for sequencing is to run them on a gel, blot to PVDF (not nitrocellulose), and stain with Ponceau Red or Coomassie Blue. Note that the maximum amount of PVDF which can be loaded is about 4 mm x 9 mm, or two bands.
Left: Loading strips of PVDF into sequencer Right: Loading liquid sample onto Polybrene treated glass fiber filter
The sample must contain only one protein, or else ambiguous data will be obtained. The N-terminal of the protein must also be unmodified; unfortunately many proteins have a modified N-terminus, which cannot be determined prior to sequencing. Peptides produced by digestion of a protein have unmodified N-termini and thus can readily be sequenced.
Our instrument can readily sequence 10 pmole of standard protein, but we suggest supplying more sample if it is readily available, because sample losses during handling often reduce the amount we receive from what people estimate. The length of sequence obtained may be limited by sample amount, but is always limited by the properties of the protein. Side reactions increase the background of amino acids and decrease the amount of amino acid released from the protein. Large proteins give more background signal than smaller proteins. Our best run was about fifty amino acids.
Edman chemistry can identify radiolabelled phosphorylation sites in peptides. Other modified amino acids may not be identified but their presence inferred by the lack of data. Cysteine cannot be positively identified unless it is alkylated. We have procedures for alkylating with N-isopropyliodoacetamide and 4-vinylpyridine, which are our preferred reagents, including a procedure for alkylating proteins prior to electrophoresis and after blotting to PVDF.
Available is a document with a description of sample preparation methods including many references and an explanation of the process of protein sequencing. (192K document). When submitting a sample, please include a sample submission form .
Mass analysis of peptides and proteins
The W.M Keck Biomedical Mass Spectrometry Laboratory can accurately determine the mass of peptides and proteins. Mass determination is often used to confirm correct synthesis of peptides and proteins. Mass spectrometry determines protein and peptide masses more accurately than gel filtration or gel electrophoresis in part because the mass is measured more directly than with other techniques; for example, the mass of a peptide with a size of 2000 can be determined with an accuracy of 0.2 mass units. The instrument we use is a PE Biosystems Voyager DE-Pro with delayed extraction and reflector.
Most often, masses are determined by Matrix Assisted Laser Desorption Ionization-Time Of Flight mass spectrometry. In this technique, the sample is mixed with a matrix, commonly cyanohydroxycinnamic acid for peptides or sinapinic acid for proteins and dried. When irradiated with a nitrogen laser, the matrix adsorbs energy which is transferred to the peptide molecule which desorbs into the gaseous phase. An electric field accelerates it, and by measuring the time it takes to reach a detector, the mass can be determined. In some cases, samples are analyzed by loading into an ion trap mass spectrometer using an ElectroSpray Interface (ESI).
Samples should be at a concentration of at least 5 pmole/µl in a volatile solvent, proteins should be at a higher concentration. 
Salts and detergents will interfere, and may prevent any data being obtained; in some cases, the sample can be desalted on minature reverse phase adsorbents. The behaviour of samples is varied, and some samples give very weak signals. If proteins are heterogeneous because of ragged termini or carbohydrate or other, the data obtained will be poor.
When submitting a sample, please a sample form.
Our mass analyses are performed on a PE Biosystems Voyager DE Pro with delayed extraction and reflectron optics.
Identification of modifications in proteins
Protein sequencing, especially by mass spectrometry, can identify modifications in proteins. However the process is usually more complex than routine sequencing.
The modification is usually only a small fraction of the total protein, so finding the modification can be difficult without further information. Knowing what modification is sought makes identification by mass spectrometry feasible. However even with this data, there is the problem that a peptide containing the modification may not be found in the initial digest of the protein. As with other samples, we recommend that investigators consult with Dr. Sherman or Shannon before starting an experiment to identify a modification in a protein.
Identification of phosphorylation sites by mass spectrometry without prior knowledge of the site is expected has a 1 in 10 chance of success. Factors making identification difficult are: low stoichiometry of phosphorylation making a phosphopeptide a few percent of the total; chromatographic behavior causing the peptide not to be seen; reduced sensitivity of detection of phosphopeptides. Our recommended approach is:
- separate tryptic peptides from a radiolabelled protein on 2D-TLC. This method is commonly used to show how many phosphorylated peptides are present.
- extract peptide from TLC plate
- Edman sequencing of labelled phosphopeptide to identify site of phosphorylation (no amino acid sequence obtained)
- use position of phosphorylation relative to trypsin cleavage site to identify candidate phosphopeptides
- Perform a tryptic digest on non-labelled material and analyze with the mass spectrometer set to ignore the majority of peptides and look only for candidate peptides.
A paper using this procedure is: Roof, R.W., Haskell, M.D., Dukes, B.D., Sherman, N., Kinter, M., Parsons, S.J. (1998) Molec. Cell Biol. 18 7052-7063
2-D electrophoresis
The Biomolecular Research Facility no longer performs 2-D electrophoresis as a service, but retains equipment which may be useful for those running gels.
The facility has a scanner for Coomassie and silver stained gels, and films; Bio-Rad FX fluorescent scanner with settings for common fluorescent stains. Stains we have scanned for are Sypro Ruby, Pro-Q Diamond phospho-protein stain, Cy3, Cy5. Images can be analyzed with Bio-Rad PDQuest software; comparison of images can be time consuming, commonly because of streaks and faint spots. The images can be converted to TIFF files for use with other programs.
A spot cutter can cut out spots, and lanes, from gels as an alternative to manual cutting. For large numbers of spots, the spot cutter may be more convenient than manual cutting. The spot cutter may also be more convenient than manual cutting for selecting Sypro labeled spots because it has a short wavelength UV lamp. The spots can be chosen from an analyzed image, or by manual selection on screen. There is a charge for using the scanners.
Digestion of proteins
The Biomolecular Research Facility performs enzymatic and cyanogen bromide digestions of proteins, separates the resultant peptides and sequence them by mass spectrometry or Edman sequencing.
The most common means of digestion are with lysyl peptidase, which cuts after lysine residues; trypsin, which cuts after lysine and arginine residues and cyanogen bromide which cuts after methionine. These digestions have been reliable.
Prior to digestion or sequencing, it is preferable to reduce and alkylate the protein to eliminate disulfide bridges, and hence cross linked peptides and protect cysteine; without alkylation, cysteine cannot be identified during Edman sequencing. The preferred alkylating reagent for Edman sequencing is N-isopropyliodoacetamide: iodoacetate and iodoacetamide give derivatives which are not easily distinguished from other amino acids during Edman sequencing. We can alkylate proteins after transfer to PVDF.
Separation of proteins and peptides
The Facility performs peptide and protein purification by reverse phase high performance liquid chromatography. Commonly purifications are performed on a small bore instrument which uses 2.1 mm and 1 mm diameter columns. Compared to the common 4.6 mm diameter columns, the small columns give higher sensitivity and supply the sample in a small volume which is suitable for Edman sequencing or mass determination. However larger columns can be used for larger samples. When necessary, peptides from a digest are rerun on a different column or in different solvents to separate co-eluting peptides. The Facility also has an FPLC for ion exchange separations of proteins.
Amino Acid Analysis
Amino acid analysis determines the amino acid composition of a peptide or protein. It is also one of the most commonly used technique for quantitating proteins and peptides that does not depend on specific structural features of the polypeptide, like the conventional dye-binding methods. Amino acid analysis is often employed to asses coupling of peptides to carrier proteins like KLH; the quantitation is aided by incorporating a non-naturally occurring amino acid, such as norleucine or ß-alanine, in the peptide.
To start amino acid analysis, the polypeptide is normally hydrolysed in 6N hydrochloric acid vapor for 24 hours at 110°C. The free amino acids are then derivatized either with phenylisothiocyanate to produce phenylthiocarbamyl (PTC) amino acids. The derivatized amino acids are separated on a C18 reversed phase column with an acetonitrile gradient in sodium acetate buffer.
Sample Requirements
We need 2 to 10 µg of purified protein or peptide sample, preferably in a volatile solvent or solid. However small amounts of non-volatile salts can be tolerated. We encourage potential users to include a buffer solution for control.
Limitations
During acid hydrolysis, asparagine and glutamine are deamidated to aspartate and glutamate, respectively, rendering the acid and its corresponding amide indistinguishable. Tryptophan is destroyed under the standard hydrolysis conditions, and so is cysteine although to a lesser extent. Therefore special hydrolysis conditions are required for these two amino acids and they need to be carried out separately.