1、What is the advantage of Nano LC-MS for protein ID?
2、For protein identification, do we need to concentrate a sample before running a SDS PAGE?
3、How to concentrate a protein sample before loading on gel?
4、What type of gel should I use for gel electrophoresis?
5、What type of gel staining method should I use?
6、How to cut a protein band out of a gel?
7、What is the advantage of Nano LC-MS in protein post-translational modification?
8、What is the minimum amount of protein that you need in gel slices for MS analysis?
9、Do you accept radioactive samples?
10、For De Novo sequencing service, what's your requirements on the sample?
11、For De Novo sequencing service, what are included in the final deliverables?
12、For De Novo sequencing service, how do I know the sequence is correct?
1. What is the advantage of Nano LC-MS for protein ID?
There are two major reasons that make the Nano LC-MS a much more sensitive technology than MALDI-TOF MS in protein identification. First, since both mass of the peptides and two serials of fragments of each peptide are present, a protein can be identified even with a low as one peptide from the digest. In contrast, MALDI-TOF based protein ID can only determine the masses of peptides; therefore it requires the detection of a large number of peptides to achieve high confident protein identification. Second, during MALDI based MS analysis, only very small percentage (typically 1/1000 to 1/10000) of peptide analyte can be often utilized. The combination of these two factors makes the LC-MS much more sensitive technique than MALDI-TOF based MS technique, although the throughput of MALDI -TOF based protein identification is hundreds times higher than Nano LC-MS based protein identification. Another important advantage of Nano LC-MS in protein identification is its ability to analyze very complex mixture, since peptides in a sample is separated by HPLC before MS and MS/MS. As a result, even a minor fraction in the protein mixture can be identified if one or more peptides from this protein are not completely buried by other peptides during entire LC separation. In contrast, peptides mixture is not separated during MALDI-TOF analysis, so it is almost impossible to identify a protein that is a minor fraction in protein mixtures, and it is very difficult even to tell whether some proteins are the missed during MALDI-TOF based protein identification. Such problem become very serious when SDS-PAGE instead of 2D gel is used in sample preparation since the co-migration of several proteins in one gel band is often the rule not exception in most microscale protein purification procedures.
2. For protein identification, do we need to concentrate a sample before running a SDS PAGE?
It depends! If a protein sample is in a concentration around 100 fmole/μl, loading 10 μl per lane should result in about 1pmol protein per lane. A protein band of >1 pmol can be visualized by several type of stain (Coomassie colloid, Zn, Cu, etc) and identification of protein in such a gel band is often not very challenging to us. However, It is often the case that the volume of a protein sample prepared by a micro purification procedure is much larger than that can be loaded for gel electrophoresis. If the total amount of proteins in such sample is very low, concentrating the sample to a volume that can be loaded to 1-3 gel lanes (10-30 μl total) is probably the best way to increase the chance to visualize proteins on gel and to identify protein by us.
3. How to concentrate a protein sample before loading on gel?
Traditional methods for concentrating protein such as membrane dialysis, TCA precipitation and membrane filtering are often not applicable in micro purification procedures due to large percentage of sample loss. Drying down protein samples will lead to high concentration of salt any other chemicals that may interfere with gel electrophoresis. Based on our experience, we suggest a simple and rapid method in which proteins are bound to some reverse phase beads packed in a micro-column, washed to remove salt, and elute with SDS loading buffer (with or without boiling) before loading on gel. We have been using GeneClean from Stradegene and R1/R2 from Applied System and each method offers a good sample recovery. However, since each protein has its unique properties and different purification procedures may result in different samples, researchers are strongly encouraged to determine and optimize their best sample concentrating procedures. In principle, the way of concentrating sample will have no effect on the subsequent MS protein identification steps for a gel separated protein. So the effectiveness of a sample concentrating method can be easily judged by sample recovery rate and behavior of such sample during gel electrophoresis, which can be monitored by running a SDS PAGE loaded with pre-concentrated and post-concentrated samples at the same amount.
4. What type of gel should I use for gel electrophoresis?
Many type of polyacrylamide gels are compatible with MS analysis, including traditional Tris-Glycine gel, or many brand commercially available pre-cast gels such as Novax NuPAGE Bis-Tris and Tris-Acetate gel. For protein identification, we have not observed any significant effect resulted from different percentage of a gel or a gradient gel, or different thickness of a gel. However, we suggest not using Tricine gel for protein identification purpose based on our experience.
5. What type of gel staining method should I use?
The gel can be stained by many different methods, including Coomassie Blue staining, colloidal Coomassie staining, Cu-staining, Zn-staining, fluorescent dye staining and silver staining. Traditional Silver staining is not compatible to mass spectrometric analysis, but several modified silver staining procedures which eliminate the use of glutaldehyde based sensitizer claim to be more compatible to MS analysis. There are several commercially available silver staining kits which all claim to be MS compatible. However, based on our experience, the yield of peptide from in-gel digestion of a "MS compatible" silver stained gel band is still much lower than that from a colloidal Coomassie stained gel band with same amount of protein. Although we do accept silver stained gel samples, we encourage our customers to make effort to purify enough protein sample for using colloidal Coomassie, Cu or Zn staining (its detection limit around 10 ng), and use silver staining when purifying more protein is too difficult. The effort to optimize a purification procedure are often paid back greatly in long run since it often lead to much high success rate in identification of these protein at much high confidence level, and such experience will be important to future works.
Also, please be careful to avoid contamination in this step. Contact with bare hands or use of dirty containers or contaminated reagents may lead to failure of identification!
6. How to cut a protein band out of a gel?
A great care should be taken during cutting a protein band out of a gel! First, take all precautions to avoid direct contact of a gel with a bare hand or any potential dirty surface, such as a light box, a razor blade, or a used container. You may want to ware a clean gloves, put a gel on a clean plastic film such (such as Sara Wrap) when it is placed on a light box, and use clean a razor blade and a gel container. Please keep in mind that even a tiny piece of human skin may contain keratins that is hundred times outweigh the protein in that gel band. Second, the size of gel slice to be cut out should be close to the size of that protein band. Too mall gel size means loss of precious sample, and too large gel size may lower the yield of peptides from in-gel digest. After a protein band is excised, carefully transfer it to a clean 500 μl Eppendorf tube. Cap the tube, label it; and wrap it with parafilm to prevent the gel piece from drying (you may also add ~10 μl 1% acetic acid/water to prevent it dry). If you need to cut several bands out, do it one by one to avoid mislabeling of these gel bands. Such a mistake will be very difficult to track down.
7. What is the advantage of Nano LC-MS in protein post-translational modification?
付款方式:Studying protein post-translational modifications, such as phosphorylation, glycosylation, methylation, acetylation, ubiqitination, and N-terminal and C-terminal processing, is often a essential step towards the understanding the function, regulation and interaction of a protein at the molecular level. Characterization of protein post-translational modifications is also very important part of drug discovery process, since almost every drug target are post-translationally modified in vivo, and post-translational modification is the major function of major drug target classes, such as protein kinase, protease, protein phosphatase, and histone deacetylase Moreover, in the development of a therapeutic protein drug, a recombinant protein has to be fully characterized for the regulatory approval. Mass spectrometry and proteomic technologies provide the most powerful tools to date in characterizing protein post-translational modifications. Since every type of protein modification accompanies a definite mass change of protein, mass spectrometry is naturally the most versatile tool in analyzing of protein post-translational modifications. It also provides superior sensitivity, quantitativeness, specificity and reliability in characterizing many types of protein post-translational modifications. With all these advantages and potentials, mass spectrometric-based proteomic technique is replacing the radioactive labeling technique to become the method of choice in studying protein post-translational modifications.
8. What is the minimum amount of protein that you need in gel slices for MS analysis?
Since we use the Nano-LC-MS technology for protein ID, There is a very good chance that we can identify a very faint coomassie stained band. I can't guarantee 100% success since there is a possibility that a faint band is actually optical illusion instead of a real protein band. However, we do give our quality guarantee (free of charge if a ID fails) to all coomassie stained samples, even for an optical illusion.
Another advantage of Nano-LC-MS/MS technology is its ability to identify proteins from even a complex mixture. It is not rare for us to identify more than five proteins in a single gel band or more than 30 proteins in one solution sample.
9. Do you accept radioactive samples?
Thank you for your interest in our service! Unfortunately, we do not accept radioactive samples.
10. For De Novo sequencing service, what's your requirements on the sample?
>95% purity. >500ug protein.
11. For De Novo sequencing service, what are included in the final deliverables?
We will provide the sequence, modification and coverage of each chain.
12. For De Novo sequencing service, how do I know the sequence is correct?
The sequences are based on data from mass spectrometry. Based on our experience, the sequence we reported to our customer are more than 99% correct.