Proteomics Sample Submission
Collaborators submitting samples for the first time should schedule a meeting with the Core Director to discuss the project. The Mass Spectrometry Facility is able to handle both gel-separated proteins and protein samples in solution, such as immuno-precipitates. For gel-based separation, we strongly recommend commercial pre-cast gels (from Bio-Rad, Invitrogen etc.) and staining with colloidal Coomassie Blue (see protocol below). Other procedures can be accommodated after consultation with Core personnel.
For solution-based analysis, sample volumes should be minimized and detergent concentrations should be kept low. For database searching, we need to know the species (human, mouse, etc.) of the source of the proteins. Also, please inform us if you expect modifications (e.g. phosphorylation, ubiquitination) or if any isotope labeling is present.
Database searches are generally carried out on two different search engines, Sequest and Mascot. The results will be sent to investigators as interactive Scaffold files or in pdf format.
- Proteomics Samples (gel-based)
The preferred staining protocol utilizes colloidal Coomassie Blue (e.g. Invitrogen Simply Blue Safestain). To avoid contamination, gels should be handled under extremely clean conditions. All staining dishes need to be cleaned before use. During staining and wash steps, the dishes should be covered. Touching the gels, even with gloved hands, should be avoided. The solutions should be fresh (check expiration date)! After the last destaining step, the gel should be brought to the Mass Spectrometry Facility in a covered dish. Gels should never be placed on a scanner (even when using Saran Wrap to protect it)! If documentation is required, we will take a picture with a digital camera while the gel is in our sterile hood.
The described staining protocol will visualize proteins at the > 50 ng level. Visual bands are usually identifiable but we often obtain good results even below this level.
- MudPIT Analysis (in solution)
This type of analysis refers to a multi-dimensional nano-LC separation of a trypsin digested protein mixture. A sample in solution is reduced and alkylated at cysteine residues before enzyme digestion. This is followed by a clean-up on a C-18 cartridge to remove salts and reagents. The resulting highly complex peptide mixture is loaded onto a tandem capillary column packed with strong cation exchange beads and reversed-phase material.
Samples to be analyzed this way (typically eluted from affinity beads) should be in a small volume (< 100 µl). Certain detergents, such as SDS, should be avoided and others, such as NP-40, kept to a minimum. Best results are obtained for total protein quantities on the order of 100 µg.