Chesterfield Building
Suite 320
701 W. Main Street
Durham, NC 27701
Visit our Sample Submission page
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.
- Following the meeting consultation, a DPMCF member will send you submission link from PAMLIMS.
- Coordinate sample delivery with proteomics_metabolomics@duke.edu
Although we have workflows for sub-microgram quantities, for an in-solution digestion and analysis we ideally ask for at least 5ug 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 250ng 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.
Please visit our Sample Submission page for instructions on sample submissions and deliveries.
Depending on the workflow, we can return data in Excel format, Skyline Proteomics Software and Scaffold Software.
Tutorials:
We aim to upload the data to the user's online data repository within 2 week of sample delivery for qualitative inquiries and up to 3 weeks for quantitative inquiries. 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.
Gel bands
Samples should be stored at 4o C in either 50 mM AmBic or water, in a 1.5 mL eppendorf tube.
Solutions
Samples can be in a wide range of "typical" buffer components including various salts, detergents, chaotropoes, etc. The DPMCF has several strategies of removing non-compatible components prior to LC-MS analysis. The most important factor, however, is giving 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
Working directly from solution in <100uL of elution volume 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.
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 localization algorithm with Spectronaut 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.