First time users, non-affiliated researchers, those interested in protein modifications and anyone wanting to submit large numbers of samples at once (more than 15) are urged to contact the facility prior to sample submission. We recommend the use of coomassie blue stain over silver, but if you use silver stain make sure to use a mass spec compatible silver stain protocol or kit. Please provide an image of the gel and indicate the bands or spots that are to be analyzed.
Please fill out the Sample Submission Form and answer the questions listed. The more information we have about the nature of the samples the better we will be able to serve you. Please provide a University of Pittsburgh billing number or a P.O. order form containing a P.O. number, the amount of money on the P.O. number, and the billing address for sending the invoice. Samples will not be analyzed unless your P.O. form is received. All P.O. numbers should be made payable to the University of Pittsburgh. Results will be sent via e-mail. All the data for each sample will be contained in an html link. For the fastest turnaround time please submit samples on Monday. We only process one set of samples each week starting on Monday. Samples submitted on Monday will be ready the following week on Monday.
The detection limit of proteins from SDS-PAGE gel bands through the use of LC/MS/MS is in the femtomole concentration range or on a weight basis in the low nanogram range. This is also very similar to the detection limit of proteins stained with silver and sypro ruby stains. Colloidal coomassie stains typically have detection limits between 10ng and 20ng and coomassie brilliant blue has a detection limit near 50ng. The detection limits of these stains will be dependent on many variables such as the thickness of the gel and the width of the lanes. While the detection limit of protein staining is on a weight basis the detection limit of protein with the mass spectrometer is on a molar basis; therefore the higher the molecular weight, at the same mass, the higher the detection limit will be for the mass spectrometer. For example 1ng of a 20kd protein is 50fm, while 1.0ng of a 200kd protein is only 5fm. Both proteins will have similar stain intensities, but there is 10 times less protein on a molar basis from the 200kd protein. In general the detection limit for protiens from gels will be 2-5ng for proteins less than 75Kd, 5-15ng for protein between 75-150Kd, and 15-25ng for proteins from 150-250ng. Protein stains also detect total protein, while the mass spectrometer detects proteins individually. It is very common for gel bands to contain several and sometimes dozens of proteins.These proteins will result in a band on the gel that is detectable with silver stain (i.e. a total of 5ng of protein), but there may not be any single protein in the band that would be detectable with the stain by itself and therefore most likely not detectable by the mass spectrometer. For these reasons we encourage the use of coomassie stain. It is a good strategy if you are not sure if you will be able to detect your protein with coomassie to run a small amount of your sample (1-5%) on a gel and use silver stain. In one of the lanes of the gel you can run a known amount of protein. We would suggest running 5 to 10ng Phosphorylase B (Sigma-Aldrich P4649) or B-Galactosidase (Sigma-Aldrich G8511), or another protein that you have available. The stain intensity of your sample and the known proteins should indicate whether you will be able to detect the rest of the protein with coomassie or if you will need to stain the remaining protein with silver.
Exemplary Workflows in Proteomics
Mass spectrometry and redox proteomics: Applications in disease
D. Allan Butterfield1, Liqing Gu, Fabio Di Domenico, Rena A.S. Robinson
Mass Spectrom Rev. 2014 Jul-Aug. PMID: 24930952
Post-translational modifications are, among other things, indicators of the redox state of a cell. Several redox markers can be found appended to proteins, such as carbonylation, glutathionylation, 3-nitrotyrosination, and 4-hydroxynonenal formation. These markers can be selected for upstream of mass spectrometry to yield analyses of oxidation state as related to cells or specific proteins. Methods include 2D Western blotting, 2D DIGE, and avidin affinity chromatography for carbonylation, and hydrazide beads for 4-NE. Specific reagents can also aid in detecting these modifications, such as dansylhydrazide, which increases the ionization efficiency of carbonyl groups.
Decreasing the amount of trypsin in in-gel digestion leads to diminished chemical noise and improved protein identifications
Hu M, Liu Y, Yu K, Liu X
J Proteomics. 2014 Jun 28. PMID: 24984109
In-gel digestion with trypsin is a common method of yielding peptides from proteins separated or cleaned via gel electrophoresis. Generally, trypsin is added at a very high quantity (25-50ul of a >10ng/ul suspension), as conventionally, a high amount of trypsin is thought necessary for effective penetration of the enzyme into the gel matrix. However, Hu et al, 2014 show evidence that such a high quantity of trypsin is unnecessary for trypsin activity, reducing the concentration of the suspension from the conventional concentrations down to 1.2 ng/ul before significant reduction of digest product signal. Furthermore, the experiments suggest that protein identification capability improve with decreased concentrations of trypsin during digests, in addition to reducing contaminating signal from trypsin autolysis.