City of Hope, a NCI-designated Comprehensive Cancer Center
General Guidelines for All Samples:
1. First-time users should discuss their project and the sample submission process with the manager or assistant manager before preparing their samples. We can help you design an experiment that will best take advantage of the mass spectrometers’ capabilities, but only if we can make suggestions early in the experimental process.
2. All users should contact us before sending or bringing samples, both to check our sample backlog and to ensure that someone will be there to receive them.
3. All samples require a guarantee of payment. On-campus users may supply a City of Hope account number. Off-campus users without an established record of timely payment must provide a purchase order or credit card number.
4. All submitted samples must be accompanied by a filled sample submission form. You can download this form on this website.
Specific Guidelines for Gel Bands:
What to provide:
Each gel band that you submit should be cut out using a disposable cutting tool, placed in a clean micro-centrifuge tube, and legibly labeled with a unique name. If you give your samples descriptive names like “band 1” or “44 kDa”, please add some additional information, such as an experiment name or notebook page number, to distinguish it from similarly named samples. When submitting a large batch of gel bands, you may place them in the wells of a 96 well microtiter plate, together with a written list of which samples are in which well. In addition to your samples and the sample submission form, please include:
1. A clearly indicated gel blank for each physical gel. The gel blank should be from a region of the gel that has no protein in it, either from between two lanes or (ideally) from a lane that was loaded only with loading buffer. Regions from any gel lane that had protein loaded in it must not be used as blanks, even if they lack any apparent stain. The gel blank will be analyzed as a control at no additional charge.
2. A picture of the gel with the positions of each excised gel band indicated.
3. Any other information you want us to consider when analyzing your sample, such as the organism from which the protein is purified, preparation method, identity and sequence of proteins you expect to find, etc.
Remember that your gel is being run as a preparative technique for further analysis, not as a final analytical method. The mass spectrometers are as sensitive to contaminant proteins as to target proteins, so you must handle the gel in a way that avoids contamination. Known causes of contamination include:
1. Contact with hair or skin. Keratin is by far the most common contaminant. Wear gloves and a lab coat whenever you work with a gel. Wear a hair net if you have long hair. Carry out as many procedures as possible in a laminar hood.
2. Contact with latex gloves. Use nitrile gloves instead.
3. Use of autoclaved labware. Use plastic labware straight from the manufacturer’s packaging without any additional treatment.
4. Use of contaminated containers. Never stain a gel in a container that has been used for Western blotting. The best choice is to use disposable plastic containers.
5. Use of old razor blades or dirty cutting surfaces when excising gel bands. Use a new razor blade, scalpel, or disposable gel cutter and either a disposable plastic surface or a freshly cleaned glass surface to cut on. A razor blade or scalpel should be cleaned between each gel band you cut out.
Specific guidelines for small molecules (metabolites, lipids, phospholipids, etc)
These will vary depending on the chemical nature of your analyte and the purpose of the analysis, and will be determined during your consultation with the manager. An internal standard will be required for quantitation. For absolute quantitation a labeled compound (stable isotope) with identical structure with your analyte must be used. Otherwise, relative quantitation can be accomplished using an internal standard with similar structure as the analyte. You must provide pure samples of all analytes for absolute or relative quantitation. Quantitation of all compounds not previously analyzed in the MS facility requires method development prior to analysis. A discussion with the manager of the facility for scientific and technical assistance it is required for all analyses of this type. Any changes in the project after the method was developed may require additional method development and associated costs. For example: you decide to add additional analytes that may require different chromatographic conditions than the analytes for which the method was initially intended.
Specific guidelines for nanospray analysis of small molecules and peptides
All nanospray samples must be pre-purified and free of buffers (such as phosphate). A negative control (blank) may also be required. Please contact the facility for sample specific information.
Specific guidelines for oligonucleotides
All oligonucleotide samples submitted must be HPLC purified and desalted using a cation exchange resin. Contact the facility for more information. Alternatively a large batch of samples can be accepted undesalted. The batch must consist of at least 20 samples. These would be analyzed by LC-nanoESi with online desalting on a PRP column using 2-10mM TEA.
Bringing or Sending Samples
If you work at or near City of Hope, feel free to bring your samples in person. Doing so will guarantee the best possible handling. Please remember to contact us before bringing the samples so we can ensure that someone is there to receive them.
If you are not able to bring your samples in person, you should send them using an overnight delivery service such as Fed Ex, UPS, etc. Please package your samples with freeze packs or dry ice to keep them cold, and be sure to label the packages “freeze on arrival” so that our receiving department keeps them cold after delivery. Avoid sending samples on Thursday or Friday to minimize the chance that the samples will remain undelivered and at room temperature over a weekend. Please email us the sample information, including tracking numbers, once you have sent it.
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City of Hope strongly supports and values the uniqueness of all individuals and promotes a work environment where diversity is embraced