Protein identification by mass spectroscopy
N-terminal protein sequencing by Edman degradation approachProtein identification by tandem mass spectroscopy
Genosphere Biotech. provides positive protein identification services using nanoflow LC/MS/MS from one- or two-dimensional SDS-PAGE gels samples. A typical analysis starts with in-gel proteolytic cleavage of the protein(s) sample followed by extraction and mass spectrometric analysis of the resulting peptides. Tandem spectra are further acquired after individual precursor peptide ions are isolated and fragmented thus obtaing peptide sequence information.
- Fig 1. Protein identification by tandem mass spectroscopy. Typical tandem MS experiment showing B-type and Y-type daughter ions
- Click on figure to enlarge
| Catalog No. | Service description |
| F201 | Protein identification: gel - Coomassie |
| F202 | Protein identification: dried gel-Coomassie |
| F204 | Protein identification: 1-3 proteins mixture in solution |
| F207 | High sequence coverage - trypsin |
| F208 | High sequence coverage - 3 proteases |
N-terminal sequencing: Edman’s approach
This procedure involves the sequential (i) chemical modification of the N-terminal residue, through its amino group with phenylisothiocyanate (PITC), (ii) removal of NH2-derivatized amino-acids from the N-terminal, and (iii) reverse phase HPLC analysis with UV detection at 270 nm.
Comparison of the successive elution profile to that of a standard set of adducted amino-acids allows for sequence calls (Fig.1).
- Fig 2. HPLC elution profile of a standard mixture of 19 adducted amino-acids providing standard retention times. Several by-products, indicated by an X, are formed during the Edman degradation process.
- Click on figure to enlarge
Tandem mass spectroscopy protein analysis
Sample form and amount
We accept bands or spots that are clearly visible with Coomassie blue and that are cut out from regular 1D or 2D SDS-PAGE gels. Amounts of 10-50 ng for average size proteins is the low end of the amount requirements. Therefore, all visible Coomassie-stained bands contain enough material for reliable analysis. We recommend to avoid silver staining since it sometimes interferes with sample extraction from the gel. We have been successful with sypro orange dye.
Note: Dried gels We now accept protein bands that are cut out from dried, cellophane covered old gels that were stored at room temperature (up to 15 years old).
Edman degradation sequencing
Sample amount
We require a minimum 20 pmol of blotted protein. 50 pmoles is very comfortable and generate unambiguous data.
| Protein size | µg for 20 pmoles |
| 10 kDa | 0.2 |
| 25 kDa | 0.5 |
| 50 kDa | 1.0 |
| 75 kDa | 1.5 |
| 100 kDa | 2.0 |
| 125 kDa | 2.5 |
Sample form
We accept PVDF blots or purified lyophilised/solubilised proteins.
Blotted onto a PVDF membrane (preferred)
The protein should be electroblotted from 1D or 2D gels and stained with Coomassie Blue (R250). Following staining/destaining, blotted membranes should be rinsed thoroughly with deionized water. The sample should be as concentrated as possible (15 to 20 pmol / lane) and several gel lanes may be loaded and submitted. The whole membrane may be submitted with a copy showing the band(s) of interest or the bands may be cut out and submitted, please indicate band molecular weight.
Protein in solution or lyophilised
30 to 150 µl of solution (H2O, Acetonitrile, Propanol). Pure lyophilised protein is also acceptable.
Protein should be free of detergent, salts and buffers. Please note that any amine-containing chemical present (e.g. Tris) will impair the adduction step.
Extra charge applies for this type of sample as we will run pre-purification at nominal fee to ensure complete removal of amine-containing impurities.
Sample purity
PVDF blots: well separated bands.
Liquid: Single purified proteins. Immunoprecipitation and affinity-column purified usually give good samples for sequencing.
In any case, a mandatory additional column purification step is applied to all incoming solution samples.
LC/MS/MS Protein analysis
The most common reason for analysis failure is high contamination levels by keratin. Keratin is a ubiquitous hair and skin protein present on dust, equipment and lab benches. Using high grade reagents (most notably SDS), cleaning all equipment prior to use and staining, and wearing gloves at all time will help minimize contamination.
The choice of protein stain greatly influence the success of the MS analysis. We strongly recommend the use of Coomassie Blue staining as it is fully compatible with the analysis protocols. For more sentitive staining, one may use fluorescent dyes (eg. sypro, ruby). Typically a visible comassie stained band will contain enough material for a reliable analysis. We recommend to avoid silver staining since it was shown in many instances to interfere with gel extraction.
Sequencing by Edman degradation
Peptides & proteins with blocked N-terminal amino groups (e.g. acetylated, formylated) will not sequence by Edman degradation.
High molecular weight proteins (over 75 kDa) may prove difficult to be analyzed. In our experience, the foremost reason is sample quantity and / or purity. However, reasonably pure protein bands and comfortable amounts will allow for unambiguous sequence calls. Please contact us before sending your sample.
Unmodified Cys and glycosylated residues will give a blank cycle (i.e. residue is cleaved off the peptide chain but is not detected by HPLC at 270 nm).
Sample quality is critical. Any impurity that interfers with Edman’s chemistry (mainly amine-containing chemicals) will lead to poor results.
Protein identification by nanoLC-MS/MS analysis
Pricing
For simple 1D or 2D gel spots / bands, a set up fee applies that includes sample preparation, trypsin digestion, peptide extraction, nanoflow LC MS/MS experiment, data collection, data analysis and database search. Please contact us for an offer.
Submitting your sample
A sample submission form in pdf format is available for download: here
Please fill and send the form with an image of your gel clearly showing the spots or bands that were cut along with the samples. Please include a copy of your Official Purchase Order (mandatory).
Shipping is simple: cut the band/spot of interest, place it in a 1.5ml microfuge tube and mail in a regular padded envelope (no ice needed).
Please send at room temperature using express courier or postal services to:
GENOSPHERE BIOTECHNOLOGIES
Bio-analysis Services
2 rue des Gravilliers
75003 Paris
FRANCE
tel: 01 42 71 70 21 (local dialing)
For a faster processing of your order and safer tracking of your samples, please advise our Customer Services of your sending a sample with courier airway bill number if available: info@genosphere-biotech.com.
N-terminal sequencing by Edman degradation
Pricing
A set up fee (minimum charge) applies that includes the first 5 residues. The latter is sufficient for confirmation sequencing. If you are studying an unknown protein and wish to search databases for homologous sequences we would then recommend at least an additional 5 residues for useful database search results. Please contact us for an offer.
Sending your sample
A sample submission form is available for your convenience: here
Please send your PVDF membrane(s) or tube(s), clearly labelled and wrapped with parafilm in a padded envelope. Include your Institutional Purchase Order (mandatory) and the sample submission form or a letter describing your sample(s) and indicating the number of residues you wish to sequence. Should you require a database search, please indicate protein details (species, suspected family, etc.).
PVDF samples: copy marking band(s) of interest, approximate size, amount loaded.
Solution samples: sample name, approximate size, total amount (µg), concentration (µg/µl), volume (µl) and solution content (H2O, buffers…).
Please send at room temperature using express courier or postal services to:
GENOSPHERE BIOTECHNOLOGIES
Bio-analysis Services
2 rue des Gravilliers
75003 Paris
FRANCE
tel: 01 42 71 70 21 (local dialing)
For a faster processing of your order and safer tracking of your samples, please advise our Customer Services of your sending a sample with courier airway bill number if available: info@genosphere-biotech.com.