Proteomics and Metabolomics FAQs | Duke University School of Medicine

Where are you located?

Chesterfield Building
701 W. Main Street
Durham, NC 27701

I would like to submit a sample for analysis. What do I need to do?

Schedule a project meeting by emailing proteomics_metabolomics@duke.edu to connect with appropriate scientist to discuss your experimental goals and sample preparation before beginning your experiments for analysis by mass spectrometry.

If you have not previously done so, you will need to create an account on our single sign-on system.
- If you are a Duke employee, you can use your NetID and password to create an account.
You will also need to create an Express Repository account so we can deliver your data.
Email a proteomics_metabolomics@duke.edu with your NetID to enable digital sample submission.
Finally, create and submit your samples on the Proteomics and Metabolomics Sample Submission System.
- Please print out the submissions page to bring with you when you drop off sample

How much protein do I need to submit for protein identification or quantitation?

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 complete an analysis with slightly 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.

How do I arrange to drop off a sample? Do I need to make an appointment?

Please visit our Sample Submission page for instructions on sample submissions and deliveries.

When would I perform solution-versus gel-based analysis of my lysate or immunoprecipitate?

For label-free quantitative analyses, we typically use in-solution digestion to limit the amount of sample-to-sample variability. We also recommend analyzing in-solution digests to obtain a qualitative "parts list" of the most abundant proteins in your sample. This method helps avoid bias due to extraction efficiency from the gel, and better raw sensitivity for samples where very little sample is available. We can perform 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) qualitatively identifies the composition of a particular gel band(s) following immunoprecipitation or protein purification. Several other scenarios show that gel-based analysis can be useful. For example, GeLC can exclude one or more high-abundance species from the analysis, which is useful in co-immunoprecipitation studies where the bait protein has higher levels than its interactors and would otherwise interfere with detecting 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 for increased proteome coverage. We have also used SILAC in combination with GeLC to quantify proteome-wide changes in response to cellular stimuli.

What software do you use to return data?

We use Skyline Proteomics Software and Scaffold Software.

Tutorials:

What is the expected turnaround time for routine analysis such as protein identification from a 1D gel band or a sample in solution, PTM mapping, analysis of interacting from an IP, etc.?

We aim to upload the data to the user's online data repository within two week of sample delivery. Once samples have been entered to our online sample submission system, our staff constantly tracks the status of each sample (including number of days since submission). 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.

What is SILAC and how would I utilize it?

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 to quantify protein-level changes in cell lysates and immunoprecipitates as well as to quantify 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.

What cells are amenable to SILAC, and how do I prepare media?

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.

In what buffer can I submit my sample?

Gel bands
Samples should be stored at 4^oC in either 50 mM AmBic or water, in a 1.5 mL eppendorf tube.

Solutions
Samples should 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

Buffers: PBS, Tris-HCl, HEPES, MES, MOPS, etc.
Detergents: Triton X 100, NP40, SDS
Other solvents: TCA, EtOH, MeOH, formamide, acetone

I have an immunoprecipated and/or tagged protein for which I would like to qualitatively map unknown phosphorylation sites. Can I work directly for a SDS-PAGE gel, or does the sample need to be in a sample?

Working directly from solution is the preferred method; although, samples can be analyzed directly from an SDS-PAGE gel band, if needed.

*Note: phosphatase inhibitors such as KF and sodium orthovanadate are compatible with the enrichment-LC-MS protocol and should be included during sample preparation.

I have an immunoprecipated and/or tagged protein for which I would like to qualitatively map unknown phosphorylation sites. How much material do I need, and how pure does the sample need to be?

We need a minimum of 0-15 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 phosphorylation via mass spectrometry, will you give me just the protein that is phosphorylated or the actual phosphorylated residue?

Phosphorylation analysis via mass spectrometry is done on proteins that have undergone proteolytic digestion via trypsinization (See Protocols & Reagents), so the sequence information and phosphorylation status is obtained at the peptide level. Therefore, we can at least 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 provide a statistical confidence for the localization of the phosphate on the peptide.

Does your lab require a specific SDS-PAGE gel system for downstream LC-MS analysis?

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.