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For label-free quantitative analyses, we most often use in-solution digestion because this affords the least amount of sample-to-sample variability. We also recommend the analysis of in-solution digests for obtaining a qualitative "parts list" of the most abundant proteins in your sample. In-solution digestion and subsequent analysis has the advantage of avoiding bias due to extraction efficiency from the gel, and better raw sensitivity for samples where very little sample is available. We have performed in-solution digestion and analysis of samples with as little as 0.1 ug of protein.
The most common application of SDS-PAGE, in combination with LC-MS/MS (GeLC) is to qualitatively identify the composition of a particular gel band(s), following immunoprecipitation or protein purification. There are numerous other scenarios where gel-based analysis can be useful. For example, GeLC can be used to exclude one or more particularly high-abundance species from the analysis. This is useful in many co-immunoprecipitation studies where the bait protein is present at much higher levels than its interactors and would otherwise interfere with the detection of low-abundance interacting proteins. GeLC can also be used in conjunction with 1D-LC-MS/MS analysis to provide a two-dimentional separation of your sample. This can result in increased proteome coverage. We have also used SILAC in combination with GeLC to quantify proteome-wide changes in response to cellular stimuli.
We use Skyline Proteomics Sofware and Scaffold Sofware. You can view tutorials on how to use this software below:
Skyline Proteomics Software Tutorials from the MacCoss Lab, U Washington
Tutorials from Proteome Software on Scaffold software
From the date of sample delivery, we aim to upload the data to the user's online data repository within two weeks. Upon entry of samples to our online sample submission system, the status of each submitted sample (including number of days since submission) are constantly tracked by Proteomics and Metabolomics Shared Resource staff. Depending on the number of other user samples in the queue, data is often returned for small projects (up to 5 samples) well within the two-week guidelines. Projects with a larger number of samples are highly dependent on the workflow employed and laboratory workload and are subject to timeline projection on a case-by-case basis.
SILAC stands for Stable Isotope Labeling with Amino Acids in Cell Culture. It can be used to perform relative quantitation across several samples generated from cultured cells. In a typical experiment, two populations of cells are grown in light or heavy media, where the latter contains 15N/13C-containing Arg and Lys. After cells are treated under two conditions (e.g. test and control), the samples are mixed, and this mixture is subjected to MS analysis. We have applied SILAC for quantifying of protein-level changes in cell lysates and immunoprecipitates as well as for quantifying post-translational modifications (e.g. phosphorylation and S-nitrosylation) between two samples. SILAC is compatible with GeLC/lanewalking or other types of multi-dimensional or subcellular fractionation.
Many transformed cell lines are amenable to SILAC. Several companies sell Arg- and Lys-free media, including DMEM and RPMI. Custom formulations are also available from Invitrogen. We have performed SILAC on HEK-293, A549, RAW246.7 and B35 cells.
We can provide detailed instructions for preparing SILAC media and suggest that you consult with us before purchasing SILAC reagents.
For gel bands, we prefer to receive them stored at 4C in either 50 mM AmBic or water, in a 1.5 mL eppendorf tube. For a solution submission, we prefer the sample already be in 0.1-0.5% Waters RapiGest MS compatible detergent in 50 mM Ammonium Bicarbonate. If this is not possible, please give us as many details as possible on the sample submission page. We need to know how to properly clean up the sample if it is in any other buffer, so it is critical that we know ALL OF THE BUFFER COMPONENTS. We have protocols available for the removal of many MS incompatible buffer components (some are less compatible than others), including the following:
Instructions here. Please schedule a project meeting with Dr. Arthur Moseley, Dr. Will Thompson, or Dr. Erik Soderblom to discuss your experimental goals and sample preparation before beginning your experiments for analysis by mass spectrometry. Briefly: If you have not done so previously, you will need to create an account on our single sign-on system. If you are a Duke employee, an account can be created with your NetID and password. Otherwise, you will have to create a user ID and password and use Dr. Arthur Moseley as the contact PI. In addition, you will need to create an Express Repository account (where we will deliver your data), and please email a member of the Proteomics and Metabolomics Shared Resource with your NetID to enable digital sample submission. Finally, create and submit your samples on http://proteinsamples.genome.duke.edu. Please print out the submissions page to bring with you when you drop off samples.
Although samples can be analyzed directly from an SDS-PAGE gel band if needed, it is most often preferred to work directly from solution as digestion efficiency is higher and peptides do not need to be extracted from the gel band. Note: phosphatase inhibitors such as KF and sodium orthovanadate are compatible with the enrichment-LC-MS protocol and should be included during sample preparation.
In nearly all circumstances, phosphorylated peptides exist in a much lower stoichiometry than non-phosphorylated peptides and, as such, require an enrichment procedure prior to LC-MS analysis. To perform our TiO2-based phosphopeptide enrichment protocol and be left with enough material for downstream LC-MS analysis, we ask that users provide a minimum of 10-15 ug total protein of a more pure (≥80% based on a coomassie standed SPD-PAGE) sample or 20-3 ug total protein of a less pure (≤50% based on coomassie stained SDS-PAGE) sample to maximize the chances of success.
For an in-solution digestion and analysis, we ask for 10 up for LC-MS/MS if possible, based on a BSA-calibrated Bradford assay. We can many times complete an analysis with a bit less; we aim to load 1 ug total protein onto our system for each run but require the overage due to sample losses during preparation. For gel bands, we have over 95% success rate for positive identification if the band is visible on a coomassie-stained gel. We have about a 75% success rate with silver- or sypro-stained gels because of the higher sensitivity and sample losses that occur with these staining techniques.
The analysis of phosphorylation via mass spectrometry is done on proteins which have undergone proteolytic digestion via trypsinization (see protocol LINK TO PROTOCOL PAGE ON THIS SITE), so the sequence information (and thus the phosphorylation status) is obtained at the peptide level. Therefore, at the very least, we can localize the phosphate group within a few residues. If there is only one phosphorylatable residue on the peptide (i.e., only one Ser, Thr, or Tyr) or the number of phosphates equals the number of phosphorylatable residues, then the localization is unambiguous. If there is more than one S, T or Y, we utilize the MS/MS spectrum and a scoring algorithm from the Gygi laboratory called A-Score (to be completely accurate we use its implementation in Rosetta Elucidator) to provide a statistical confidence for the localization of the phosphate on the peptide.
Although nearly any SDS-PAGE system can be utilized upstream of an LC-MS analysis, we recommend Invitrogen's NuPAGE Bis-Tris mini-gel system. A good general purpose gel covering a large MW range (6-200 kDa) is the 4-12% gradient gel (Cat. No NP0321) using MES running buffer. These pre-cast gels provide excellent resolution, fantastic staining sensitivity (10 ng BSA using colloidal blue staining kit, Cat. No LC6025), and are highly compatible with downstream LC-MS analysis. Additional product information can be found here.