Services offered by SUMS are listed below
Typical applications and links to sample preparation guidelines are provided for each type of analysis. Please contact us regarding special projects or samples that fall outside the given categories.
- Includes organic and organometallic compounds
- Confirmation of reaction products: Did my reaction give the right product?
- Location of reaction products: Which HPLC fraction is my product in?
- Rate category: Mass determination
- Molecular structure determines preferential cleavage sites in a molecule.
- Fragmentation patterns can give valuable information about molecular structure: Is the methoxy group on ring A or C of this polycyclic compound? On which part of the molecule is an isotopic label located?
- Mass Frontier, a software program designed to aid in the interpretation of MSn spectra, is available for use in Keck 328, on a SUMS data analysis workstation. Given a molecular structure, Mass Frontier will predict fragmentation patterns and pathways.
- Rate category: MSn
- HRMS can be used to distinguish between alternative molecular formulas with the same nominal mass.
- A low-resolution MS is required prior to HRMS analysis.
- Hi-res samples are typically run one day per week
- Use this template to list samples (be sure to enter the monoisotopic masses to 4 decimal places) and email it to SUMS. Submit the usual Sample Submission Form along with your samples; enter the filename of the emailed template in place of the Sample ID.
- A bulk rate is available for samples submitted in batches of 40 or more; the low-res MS screening is waived for bulk samples.
- Rate category: HiRes
- Peptides and proteins up to 60 kD are routinely analyzed
- Larger proteins require special attention to sample preparation, particularly desalting.
- Intact antibodies are analyzed by nanospray on the Q-Tof MS. Meticulous desalting (e.g. by prep HPLC, or multiple rounds of cleanup using dialysis or microcon type filters) is crucial to obtaining data on intact antibodies.
- Use LC-MS for molecular weight data from peptide and protein mixtures.
- Rate category:
Mass determination (Note: nanospray analysis of proteins on the Q-Tof will be charged at the HiRes rate)
- MS/MS of a peptide yields ion series that can be compared against the predicted MS/MS ions of a given peptide sequence.
- Can provide confirmation of identity: Was the correct peptide sequence synthesized?
- Can confirm synthetic additions or modifications: Is the tag attached to the correct amino acid?
- Generally works well for peptides with fewer than 20 amino acids
- In some cases, the source of simple deviations from the predicted sequence can be determined.
- Rate category: MSn, Custom analysis
- LC-MS or LC-MS/MS (analytical to microbore HPLC)
- HPLC coupled to MS is a powerful combination which is applicable in a wide variety of situations.
- On-line cleanup of small molecule, peptide and protein samples
- MW determination of mixture components
- Analysis of isomers or conformers
- Substrate degradation studies
- The SUMS lab possesses a limited number of reverse phase C18 and C4 HPLC columns. If unusual stationary phases or column specifications are required, you may be asked to provide a column.
- Rate category: LC-MS
- ASSAYS & QUANTITATION
- Quantitation of specific analytes, typically small molecules and peptides (AQUA), with appropriate standards
- Analysis in various matrices, e.g. serum, tissues, urine, cell culture media
- Mass range: 100-2500 m/z (mass to charge ratio)
- Sensitivity: lower limits of detection are generally in the femtomole to picomole range, depending on the nature of the analyte and the sample matrix
- Project workflows are very much compound dependent and sometimes difficult to predict; please discuss projects with Theresa McLaughlin before sending samples for quantitation
- Rate category: Custom analysis
PROTEOMICS.
Please contact Chris Adams regarding proteomic analyses.
- Rate category: Protein ID (includes digest)
Protein identifications are made by collecting LC-MS/MS data on the peptide mixture generated by proteolytic digest of the sample, then searching the MS/MS spectra against existing databases using appropriate software.
Included with a protein ID analysis:
- In-gel digestion with trypsin (cuts after lysine and arginine)
- LC-MS/MS peptide fragmentation
- Database search against a standard protein database
- Results in electronic format, including the protein hits and supporting data
- A free 100 fmol myoglobin system suitability run
Additional Work (will be charged at the project rate):
- Searches for post translation modifications
- Formatting a unique database, for example a genomic database for your species of interest
- Searching for unique features
- For digests other than trypsin the client may be asked to pay for the purchase of the enzyme; if the enzyme needs to be special ordered, delivery of results may be delayed
Protein ID does not include the following:
- Detailed peptide mapping. Sequence coverage is reported, but if complete sequence coverage is your goal you might consider a peptide mapping project
- Disulfide linkage characterization is not included in protein ID and would be performed as a project if requested
- Protein digest
- Proteolytic digestions can be carried out on both solution and in-gel samples
- Prior to trypsin digest, proteins will be reduced and alkylated with acrylamide (in gel) or iodoacetamide (solution)
- Dithiothreitol (DTT) is used as the reducing agent; if your sample is not compatible with DTT treatment, contact SUMS to discuss alternatives
- Rate category: Custom analysis
If complete sequence coverage is your goal, we would suggest digestion with multiple enzymes (up to three). In this case, three high resolution LC/MS/MS peptide maps would be run. Every effort would be made to identify as many of the peaks as possible. The average detailed peptide mapping project takes about 24-48 hours. Every effort will be made to keep the project time to a minimum. Peptide mapping is charged at the project rate.
- Rate category: Custom analysis
A good cost saving alternative to gel spot or band protein ID analysis is MudPIT analysis: multidimensional chromatography and LC-MS/MS. Quite typically the samples originate from an immunoprecipitation or some other affinity purification. A trypsin digest is performed in solution.
MudPIT can be performed on extremely complex samples, even entire proteomes. The length of the chromatographic separation depends on the complexity of the sample. MudPIT is performed at the project rate. Plan on 24 hours of project time per standard MudPIT sample. A control and an experimental sample would come to approximately 48 hours of project time.
The cost of the project includes building a triphasic MudPIT trap and packing a 60 cm capillary column for high resolution peptide mapping. Researchers will receive a Scaffold file or Mascot hyperlinks to their results. An example of typical results can be viewed here. (If you are off campus you will need to email us your IP address to be able to view this example.) The file is quite large; please allow time for the page to appear.
- Researchers are encouraged to make use of the data analysis workstations in the SUMS laboratory. The SUMS staff will provide instruction and help on using the various data analysis software programs. There is no charge for this assistance.
- Available programs include Xcalibur, MassLynx, Mass Frontier, BioWorks, Sequest, Mascot, Scaffold, ProteinLynx, ChemDraw
- Projects requiring extensive data analysis by SUMS staff will be charged an hourly rate.
- Rate category: Custom analysis
- Custom work, e.g. complex proteomic analyses, de novo peptide sequencing, method development, sample preparation, or assay development, will be charged at an hourly rate.
- Please contact SUMS to discuss projects before sending samples.
- Rate category: Custom analysis
SUMS
Stanford University
Keck Science Building
Room 328
380 Roth Way
Stanford, CA
94305-5080
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Last Modified 10.8.07
