Welcome to the Proteomics Resource Centre of the Ottawa Institute of Systems Biology (OISB). We offer proteomics services for the research community. We also participate in research collaborations. This center is supported in part by the Ontario Genomics Institute as well as user fees.

Services

Gel band analysis:
including HPLC-ESI-MS/MS analysis, Mascot search results, and data storage for 2 months.

Gel free proteomic analysis
In-solution digestion      120$ per sample

Analysis of post-translational modification:
Including 90 min HPLC-ESI-MS/MS on an Orbitrap mass spectrometer, Mascot search results, and data storage for 2 months.

Analysis of complex proteomic samples:
Including HPLC-ESI-MS/MS on an Orbitrap mass spectrometer, Mascot search results, and data storage for 2 months.

Other services:
Data analysis      150$/hour

	Extended data storage
UofO	10$/sample/year 500$/lab/year
External	20$/sample/year 1000$/lab/year

Guidelines for Sample Submission

General Information

1. We recommend the use of coomassie blue stain over silver, but if you use silver stain make sure that a mass spectrometry compatible silver staining protocol or kit is used.
2. If you wish to perform the in-gel digestion yourself, you will be held responsible if there is any damage incurred to our HPLC columns due to dirty sample preparation.
3. When filling out the Sample Submission Form , please specify the type of analysis required. Additional information about your samples will allows us to better serve you.
4. Please provide a University of Ottawa Cost Centre or valid credit card number, including the cardholder name and expiry date. Samples will not be processed unless information detailing the method of payment is received.
5. For the fastest turnaround time ( ~ 1 week) please submit samples by noon (12 pm) Monday.
6. Results will be sent via courier unless otherwise requested.
7. We do not accept samples containing radioactivity.

Background Information

The detection limit of proteins from SDS-PAGE gel bands through the use of LC-MS/MS is in the femtomole amounts or on a weight basis in the low nanogram range (1-5 ng). This is also very similar to the detection limit of proteins stained with silver and sypro ruby stains. Colloidal coomassie stains typically have detection limits between 10 and 20 ng and coomassie brilliant blue has a detection limit near 50 ng. The detection limits of these stains will be dependent on many variables such as the thickness of the gel and the width of the lanes. While the detection limit of protein staining is on a weight basis the detection limit of protein with the mass spectrometer is on a molar basis, therefore the higher the molecular weight, at the same mass, the higher the detection limit will be for the mass spectrometer. (For example 1.0 ng of a 20 kDa protein is 50 fm, while 1.0 ng of a 200 kDa protein is only 5 fm. Both proteins will have similar stain intensities, but there is 10 times less protein on a molar basis from the 200 kDa protein.)

It is very common for gel bands to contain several and sometimes dozens of proteins. These proteins will result in a band on the gel that is detectable but there may not be any single protein in the band that would be detectable with the stain by itself and therefore most likely not detectable by the mass spectrometer. For this reason we recommend the use of coomassie stain. It is a good strategy to run a small amount of your sample (1-5%) on a gel and use silver stain if you are unsure if the protein of interest will be detected with coomassie. In one of the lanes of the gel you can also run a known amount of protein. We suggest running 5.0 to 10.0 ng of a standard protein. The stain intensity of your sample and the standard protein should indicate whether you will be able to detect the protein of interest with coomassie or if you will need to stain with silver.

Staining SDS-PAGE Gels

I. Coomassie:

1. De-stain gel to a clear background so that bands can be easily seen.
2. If possible, take a picture of the gel prior to excision of gel bands and submit an image along with the sample.
3. Excise gel band(s)/spot(s) with as little excess empty gel as possible.
4. Place the gel band(s)/spot(s) into a microcentrifuge tube with some ddwater.
5. Fill out the Sample Submission Form .
6. Drop off or send samples and submission form to the facility

II. Silver:

1. Only stain the gel long enough (usually only a few minutes) to detect the bands of interest.
2. If possible, take a picture of the gel prior to excision of gel bands and submit an image along with the sample.
3. Excise gel band(s)/spot(s) with as little excess empty gel as possible.
4. Place the gel band(s)/spot(s) into a microcentrifuge tube with some ddwater.
5. Fill out the Sample Submission Form .
6. Drop off or send samples and submission form to the facility

III. Sypro Ruby:

1. Sypro is a mass spectrometry compatible protein stain with similar sensitivity as silver.
2. Since the stain is a florescent dye it can not be seen by the naked eye. Please submit a picture of the gel and indicate the bands or spots to be analyzed.
3. Excise gel band(s)/spot(s) with as little excess empty gel as possible.
4. Place the gel band(s)/spot(s) into a microcentrifuge tube with some ddwater.
5. Fill out the Sample Submission Form .
6. Drop off or send samples and submission form to the facility.

Phosphorylation

Samples for phosphorylation analysis must be at coomassie levels. The more protein you can load in the gel and the greater the amount of purified protein will increase the likelihood of identifying phosphorylation sites. Send the sequence of the protein along with the sample. We will do a theoretical trypsin digestion to determine the peptide coverage. If the peptide coverage is poor with trypsin another enzyme may be utilized.

Hints to Avoid Keratin Contamination

Keratin is the most commonly observed background contaminant for samples analyzed by LC-MS/MS. Listed below are few hints that will help you to avoid this problem:

•  ALWAYS USE GLOVES.

•  Wash all glass plates thoroughly with 70% ethanol prior to casting an SDS-PAGE gel.

•  After completing the gel electrophoresis step, disassemble the glass plates in a laminar flow hood. (Steps 4-6 should also be performed in the laminar flow hood)

•  Destain the gel in a clean container that has been rinsed thoroughly with 70% ethanol.

•  Excise each gel band of interest using a new/sterile blade.

Sample Submission Form (27 KB PDF)

Links to protocols:

In gel digestion (129 KB PDF)

Silver staining (125 KB PDF)

Contact Information:

Facility Manager:
Dr. Hu Zhou (hzhou@uottawa.ca )
Proteomics Resource Centre
Ottawa Institute for Systems Biology
Department of Biochemistry, Microbiology, and Immunology University of Ottawa Room 4501C, Roger Guindon Hall
451 Smyth Road
Ottawa, ON
K1H 8M5
Office Phone: 613 562-5800 ext. 6422