If I choose MALDI-TOFTOF for protein identification, how much sample do I need?
Answer: A good success rate can be guaranteed if the sample is a stained 2D gel dot or a band that is visible to the naked eye. Alternatively, if the sample is greater than 200 fmol (or 10 ng of 50 kDa protein), the success rate is high. For solution or lyophilized samples, a protein content of at least 100 ng is required.
For protein spot identification, 2-DE is stained with silver stain. Which silver staining reagents will affect the subsequent mass spectrometry?
Answer: Do not use glutaraldehyde in the silver staining reagent, it will modify the protein, which will affect the subsequent protein identification.
Purified protein to do glue strip phosphorylation identification, why identify to so many impurity protein?
Answer: The sensitivity of mass spectrometry is very high, and there is no way to avoid protein contamination in the experimental environment. When the target protein content in the sample is low impurity protein is highlighted. This is usually keratin contamination, and it is generally recommended to just delete the impurity proteins.
How to reduce the contamination of keratin?
Answer: The contamination of keratin comes mainly from hair and dander. Therefore, gloves and headgear should be worn during experiments and glue cutting.
Do I need to combine the glue dots?
Answer: For koji samples, one glue dot visible to the naked eye is sufficient. For silver stained samples it is recommended that 4 separate spots be combined. However, in general we recommend that silver-stained samples be identified by LC-MS-MS.
How long can samples be stored?
Answer: We recommend that freshly prepared samples be stored immediately for mass spectrometry. Silver-stained samples should be stored for a minimum of 2 months. The samples can be stored for more than a few months, but preferably not longer than 6 months.
Do you accept samples that have been digested? Do you use other enzymes besides Trypsin for enzymatic digestion?
Answer: We do not accept samples that have already been digested because we have strict internal QC controls for digestion that require us to perform the digestion. Normally we will choose a single Trypsin for enzymatic digestion, if you need other enzymes for enzymatic digestion, please contact us in advance.
How to perform identification of small molecular weight proteins?
Answer: Generally, if the molecular weight range is above 20kDa, we recommend MALDI-TOF/TOF for identification. If it is below 20kDa, we recommend LC-MSMS for identification.
Comparing the gel point identification results with the run-out bi-directional protein profiles, it was found that some of the points with the highest protein scores had molecular weights or isoelectric points that did not correspond to those on the profiles and differed significantly. In this case, how should one determine which protein has the highest confidence among several or even a dozen point proteins given in a glue point?
Answer: Some of the proteins identified by protein profiling may be homologs of the target protein or a subunit of that protein. Artificial or natural modifications or degradation of the protein can affect the position of the protein in the gum map.
In general, the higher the Mascot identification score, the higher the confidence level. It can also be combined with information such as molecular weight, isoelectric point, and peptide coverage to screen proteins with high confidence.
What is the difference between primary mass spectrometry and secondary mass spectrometry? How to choose?
Answer: The primary mass spectrometry identification method mainly refers to peptide fingerprinting (PMF). It uses mass spectrometry to accurately measure the molecular weight of enzymatic fragments and search libraries for comparison to achieve protein identification.
The secondary mass spectrometry is based on the primary mass spectrometry and then select some peptides for further fragmentation and in-depth analysis and comparison of the fragments to identify the sequence of the peptide and combine the results of PMF to achieve protein identification.
Secondary mass spectrometry is the general trend of protein identification, and even if the results of primary mass spectrometry are done now, some PMF results need to be selected for secondary mass spectrometry verification.
What if the species under study is not a model organism?
Answer: The protein can be successfully identified by referring to the nearest related model organism. If the mass spectra are good and no identification results are available, this is a novel protein, which can be analyzed and identified in depth by de-novo sequencing and other techniques.
How should I evaluate the effectiveness of mass spectrometry?
Answer: PMF generally scores over 60 (p<0.05) as a successful identification. For tandem mass spectrometry, a score of more than 60 or at least one peptide with a score of more than 30 is considered successful.
Can the gel be stored for a long time? How should it be kept? Does it have any effect on the identification results?
Answer: If the storage time is longer than one month, you can wrap it with plastic wrap and put it in the refrigerator. If it is less than one month, it can be kept at room temperature and will not affect the mass spectrometry results.
