PNAC Facility Home

Protein Sequence (Edman) analysis

Peptide Synthesis

Mass Spectrometry (samples in solution)

Mass Spectrometry (proteins in gel slices)

Amino Acid Analysis




International labs - PNAC Facility cannot accept
work from outside the UK; search the
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This restriction does not apply to the services of CCP.

Waters Maldi MicroMX ©

Contact Dr Len Packman 01223 333639 l.c.packman@bioc.cam.ac.uk

Mass Spectrometry - identifying proteins in polyacrylamide gels

Service offered
Identification is primarily by peptide mass fingerprinting using trypsin digestion and a MALDI mass spectrometer. This technique will usually identify the main component(s) but is unlikely to detect minor components. Results typically available in 2-3 days (holidays excepted). This technique gives no accurate estimation of the protein's intact mass or necessarily its N- and C-terminal locations. Superior in-depth results can be obtained from lcms/ms analysis in the Cambridge Centre for Proteomics but may take longer.

How do I know if this is the right service for my sample?
   If you want to identify the main (1-2) components of the sample and the gel band is visible by Coomassie staining then fingerprinting should be adequate.
   If you need to identify several/many components or the band is visible only through silver-staining then you need to use the lcms/ms services of the Cambridge Centre for Proteomics.

• Please read guidance before preparing your sample
• All samples must be accompanied by an official order form, detailing all relevant and requested information
• Attach a hard copy departmental purchase order (Biochemistry only - a grant code)
• Postal address. Note how to ship samples by post
• Fastest turnaround time achieved if gels delivered before 11am

MALDI Fingerprinting Prices
This includes sample preparation, digestion, sample clean-up, analysis by mass spectrometry and searching of the data against databases. Prices are per sample (gel band).

• Biochemistry Department - £40
• Cambridge University Departments - £60
• External Academic Clients - £80+VAT (no exemption); see important notes
• Industry (where capacity permits) - £120+VAT (no exemption); see important notes

Additional service options & charges
Peptide fragmentation analysis by MS/MS of unfractionated peptide mixture (not on weak bands). Prices are per sample. If this is performed because the fingerprint analysis was inconclusive, a part charge may apply in addition to the fingerprint cost.

• Biochemistry Department - £60
• Cambridge University Departments - £80
• External Academic Clients - £100+VAT (no exemption); see important notes
• Industry (where capacity permits) - £150+VAT (no exemption); see important notes

De novo sequence interpretation from MS/MS analysis and/or further general work; labour charge per hour

• All Cambridge University Departments - £40
• External Academic clients - £50+VAT (no exemption); see important notes
• Industry - £75+VAT (no exemption); see important notes

Important Sample Preparation Guidance

1. A MALDI peptide mass fingerprint can be achieved from Coomassie-stained and colloidal Coomassie-stained bands.  It may also be possibe from silver-stained bands (colloidal Coomassie stain is always preferable to silver so try that first) but the presence of keratin at this level can seriously compromise a good match score. Esi ms/ms of silver-stained bands is recommended so please approach the Cambridge Centre for Proteomics or enquire of Chemistry to use their maldi tof/tof, which has ms/ms capability.
2. Maintain a high protein:gel ratio for best results. That means preferably running thin mini-gels (1D and 2D). This minimises the volume of gel containing any protein spot. Current mass spectrometry methods have been optimised using gels stained with Coomassie R250 or Colloidal Coomassie (for better sensitivity). Most commercial Coomassie and colloidal Coomassie stains appear to be OK. If neither Coomassie approach shows your band and you choose to then try silver stain, make sure that you use the method of Shevchenko or a proprietary silver stain guaranteed as ms-compatible.
3. Keep everything very clean - human keratin (dust, fingers, hair) can be a major contaminant. Filter all solution prior to use and always wear gloves to protect the sample from you! Never handle the tubes containing your sample or the resulting gel with bare hands. Buy a new dish for staining and keep it solely for gels destined for proteomic analysis.
4. Do not over-destain your gels if using standard Coomassie. It is better to retain a faint blue background. If I can't see the band I can't cut it!
5. After staining/destaining, image the gel and wash in 10% aq. methanol. This removes the acid. For those gel stains which do not use acids or organic solvent, 10% aq. methanol acts as an important bacteriocide/fungicide so still use it. Supply the gel in minimal 10% aq. methanol (1-2ml only) in a sealed dish, bag or tube; any excess liquid is unnecessary and will compromise transport safety. Any gels left at Reception must be sealed in a bag. Archived gels, dried onto paper or dialysis membrane can be analysed although there is an increased risk of contamination particularly in low abundance samples.
6. Please do not cut the bands - leave that to me and supply the entire gel or, at the very least, the band surrounded by 1cm gel on all sides. This minimises contamination and allows me to optimise signal/noise. Do not artificially enhance the image supplied;  a better image doesn't magically make a faint band easier to see and cut. If I can't see the band I can't cut it!
7. Gels shipped by post in a Falcon-style tube require 0.1ml of liquid only to maintain humidity ; if you add more liquid the gel will be a homogenised mess when it reaches here! Follow this advice and your gel will arrive quite intact. Other methods: heat-seal gel between layers of plastic film (or use zipper-seal bag), no liquid, and ship between stiff cardboard.
8. General rule - do not work at the edge of sensitivity unless you have no choice. Better results are always obtained from decently stained bands or spots. 500fmol (few 10s nanogram) or above recommended.

Typical sample treatment protocol

  Gel bands were excised and subjected to the following treatment (30min per step, 20^oC, in 200μl 100mM ammonium bicarbonate/50% acetonitrile). 1) Reduction with 5mM tris(2-carboxyethyl)phosphine. 2) Alkylation by addition of iodoacetamide (25mM final concn.). 3) Removal of liquid then wash.  Gel pieces were dried in vacuo for 10min and 25μl 100mM ammonium bicarbonate containing 10μg/ml modified trypsin (Promega) was added. Digestion was for 17h at 32^oC. Peptides were recovered and desalted using μC18 ZipTip (MIllipore) and eluted to a maldi target plate using 2μl alpha-cyano-4-hydroxycinnamic acid matrix (Sigma) in 50% acetonitrile/0.1% trifluoroacetic acid. Peptide mass were determined using a Maldi micro MX mass spectrometer (Waters) in reflectron mode and analysed with Masslynx software. Database searches of the mass fingerprint data were performed using Mascot (http://www.matrixscience.com).
   For tandem ms/ms analysis, desalted peptides in 70%MeOH/0.2%formic acid were delivered to a ThermoFinnigan LCQ Classic ion-trap mass spectrometer using a static nanospray needle (Thermo Proxeon). Peptide masses of interest were manually selected for fragmentation using manufacturer-recommended settings. Fragment ions were matched to possible sequence interpretations using MS-Product and/or MS-Tag (http://prospector.ucsf.edu/)

Revisions: form May 04, Sep 11; prices Aug 2002, Oct 2010      Prices are continually reviewed and subject to change without notice.

Notes
You must agree to Standard Terms for Processing Samples on order form.  VAT will be payable on all external orders as exemption (zero-rate) does not cover supply of 'information' (i.e. results), even if VAT exemption is claimed by the customer. Sorry, but that's VAT law. No VAT is payable by University of Cambridge customers.

Charges will be levied as listed even if an identification is unsuccessful as significant work is involved with each sample. If no result is forthcoming through operator error or instrument failure, the charge will be waived.

VAT legislation Once inside the scope of VAT, the VAT law is such that unless something is specifically 'exempted' or 'zero-rated' it will be standard rate.  The specific bit of legislation allowing zero-rating (also referred to as medical exemption) for substances provided is in the VAT Act 1994, Sch 8, Item No 10: "The supply to a charity of a substance directly used for synthesis or testing in the course of medical or veterinary research".  This does not cover information supplied as results based on analysis of a  substance. VAT zero-rate can only apply where PNAC Facility is actually supplying a substance such as peptides.