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Protein Identification
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 FAQ Frequently Asked Questions are a good place to start, if you are planning to use our service for the first time or if you have specific questions regarding sample preparation, the analysis or report etc. Gel types Q: What gel types can I use for electrophoresis so it is compatible with MS analysis? Staining methods Q: What staining methods can I use? Protein amounts needed Q: How much material is needed to identify the protein? Spot picking methods Q: How do I cut the protein band/spot from the gel? Keratin contamination Q: What is Keratin and how do I avoid contamination of my sample with Keratin? Mass spectrometry and protein identification Q: What are the principles used in the protein identification? Q: What is the Sequence Coverage obtained in a protein identification? Q: I want to identify a protein from an organism where the genome has not been sequenced, is this possible? Q: Can you do de novo sequencing? Q: I did not get positive protein identification. What went wrong? Q: Can you identify a protein in a mixture of proteins? Q: Can you identify proteins from a Western blot? Q: Can you identify protein modifications and do more detailed protein characterization? Gel types Q: What gel types can I use for electrophoresis so it is compatible with MS analysis? A: Most standard acrylamide:bisacrylamide gels (2.6% bis) with Laemmli buffer systems work well. This includes home made gels and pre-cast gels from various suppliers, such as NuPAGE and Criterion. Homemade gels should polymerize over night before use. Other gel types and buffer systems may work as well, but do not use Tris-Trizine buffer systems. Staining methods Q: What staining methods can I use? A: You may use standard gel staining methods like Coomassie staining, colloidal Coomassie staining, Sypro Ruby, and Silver staining. The most sensitive of these methods is the silver stain. Be aware that most silver staining methods are not compatible with MS protein identification, even some commercially available staining kits that claim MS compatibility does not work properly. Successful silver stain and high sensitivity protein identification can be obtained when the referred Silver staining protocols are used. Protein amounts needed Q: How much material is needed to identify the protein? A: In general, the higher the protein amount is the better the result is. Usually, good results are obtained on clear bands/spots in a silver stained gel or a visible band in a Coomassie stained gel. This corresponds approximately to 100 femtomole – 1 picomole of protein. Good results and positive protein identifications may also be obtained at lower amounts. Proteins can be identified down to the detection limit of the silver stain (10-50 femtomole), however this is protein dependent and may not always work. Spot picking methods Q: How do I cut the protein band/spot from the gel? A: Please cut as close as possible to the edge of the protein spot of interest, with as little excess gel material as possible. You may use a razor blade or scalpel for 1D bands, or for 2D spots a glass tube or an adjusted pipette tip with the right diameter. Keratin contamination Q: What is Keratin and how do I avoid contamination of my sample with Keratin? A: Keratins are proteins naturally present in human skin, hair and clothes of animal origin (a wool sweater). Keratins are everywhere as dust; e.g. on your skin, clothes, in the air, on any open surface, glass plates, staining trays, pipette tips, and glass bottles. If Keratin is present in your sample it will be identified by the protein identification method and can in worst case obscure identification of the protein you are interested in. Therefore you should avoid dust contamination of your sample; i.e. work in a clean lab with clean surfaces, only use pipette tips and Eppendorf tubes from closed boxes, rinse equipment in clean water before use (bottles, glass plates, electrophoresis equipment, staining trays) Take good care when you cut out the bands/spots not to touch the gel with you fingers and do not touch your hair while you do it. Work fast and clean. Mass spectrometry and protein identification Q: What are the principles used in the protein identification? A: The gel spot/band is treated with trypsin that cleaves the protein after Lysine and Arginine residues and the resulting peptides are analysed by mass spectrometry. The masses of the peptides are used to query sequence databases for proteins with matching peptide masses. Some of the peptides are also fragmented by MS/MS techniques and the partial amino acid sequences obtained are used in the database query. At Alphalyse we preferably use MALDI TOF/TOF mass spectrometry because it provides:
Q: What is the Sequence Coverage obtained in a protein identification? A: The Sequence Coverage is the percentage of the protein sequence where matching peptides have been found. The larger the sequence coverage is, the more certain is the identification and the more of the sequence is confirmed by the experimental data. At Alphalyse, the sequence coverage is between 15%-90% for identified proteins. Q: I want to identify a protein from an organism where the genome has not been sequenced, is this possible? A: Yes, homologous proteins from other species may be identified if some of the peptides have identical sequences. If the mass spectrometric data are good, but no protein can be identified, then the protein is novel. Q: Can you do de novo sequencing? A: At present Alphalyse does not provide de novo sequencing of novel proteins. Q: I did not get positive protein identification. What went wrong? A: Some of the most common reasons for getting poor mass spectrometric data are:
At Alphalyse we have Quality Control on all steps and we have control samples and internal controls set of samples to monitor trypsin activity, level of keratin contamination, and instrument settings and calibration. Q: Can you identify a protein in a mixture of proteins? A: Often 2-3 proteins can be identified in a gel spot. If more proteins are present we recommend improvement of electrophoresis conditions before the spots are cut out for analysis. Q: Can you identify proteins from a Western blot? A: No, we do not analyse proteins from blotting membranes. Furthermore, Western blot samples often are contaminated with other proteins from the procedure, such as BSA or milk powder, and antibodies from the staining. Instead it is recommended to run 2 identical gels, and use 1 for the western blot, and 1 gel for cutting the spot for mass spectrometric analysis. Again is important that the proteins are well separated, because the WB will detect protein based on affinity, MS will detect based on protein amounts. Q: Can you identify protein modifications and do more detailed protein characterization? A: Some common modifications can be picked up by the database search, such as Methionine oxidation. Other modifications needs more detailed characterisation. Please contact Alphalyse by e-mail for a quotation. |
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Alphalyse A/S Unsbjergvej 4 DK-5220 Odense SØ | Denmark Tel.: +45 63106500 | Fax: +45 63106509 E-mail: info@alphalyse.com |
Alphalyse, Inc. 200 Page Mill Road, Suite 100 Palo Alto, 94306 CA | USA Tel.: (650) 543-3193 E-mail: mail@alphalyse.com |
Legal Disclaimer | Copyright 2004 © 2011 | |
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