Proteomics How Do I?

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How do I cite an ICBR Proteomics Lab in publications?


How do I cite an ICBR Proteomics Lab in publications?
For the methods used to analyze the data, please contact Sophie Alvarez. You will be provided with a description of the methods and a customized description for the special projects.
If you are considering publishing data produced by an ICBR Proteomics Lab, we would appreciate an acknowledgement sentence in the Acknowledgements. Also when published, please send us a copy so we can advertise your published paper in our ICBR newsletter and webpage.

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Mass Spectrometry and Protein Characterization

What kinds of protein gel staining are compatible with mass spectrometry and what do you recommend?
Coomassie blue staining, fluorescent staining (Sypro from Invitrogen, Deep Purple from GEHealthcare, Flamingo from BioRad) and modified silver staining (SilverQuest from Invitrogen, SilverSnap from Pierce, PageSilver from Fermentas) are compatible with mass spectrometry. However because fluorescent and silver staining are really sensitive (200 pg to 1 ng), the success of protein identification will be limited by the low amount of proteins extracted from the gel pieces. Keep in mind that the highest sensitivity of detection in mass spectrometry is around 0.1-1 pmol.
What kind of solvents should be used to resuspend peptides for mass spectrometry?
The current setup of the HPLC online with our mass spectrometers is using as buffer A: 0.1% HAc, 29.9% ACN, 97% H2O; buffer B: 0.1% HAc, 29.9% H2O, 97% ACN. The loading buffer used is the same as buffer A, plus 0.01% TFA. Please use the composition of the loading buffer to resuspend the peptides.
What buffers can I use for HPLC analysis online with mass spectrometry?
It is essential to use volatile and salt free buffers such as methanol or acetonitrile. Avoid buffers such as :TRIS, CHAPS, PBS, and HEPES. Avoid salts :e.g. Na, K, P, citrate, perchlorate. Avoid surfactants and detergents.
How do I visualize and validate protein identification in Scaffold?
Visualization of the data in Scaffold is possible by downloading the free viewer Sacffold software using this link: http://www.proteomesoftware.com/Proteome_software_prod_Scaffold_download.html.
Their website provides excellent Flash demonstrations to help you to understand the significant MS/MS data: http://www.proteomesoftware.com/Proteome_software_prod_Scaffold_tour.html.
The recommended criteria for a significant protein identification based on publications requisitions are:
  1. At least 2 unique peptides per protein identified
  2. Each peptide showing a probability higher than 95% (about a Mascot Ion Score higher than 30)
How do I validate a post-translational modification?
To validate a post-translational modification, the fragmentation pattern must be visually validated. The y or b ions series must cover the amino acid with the post-translational modification. The fragmentation table in Scaffold can help you to check the coverage of the sequence by the y and b ions. For a phosphorylation, first the peptide size must be shifted by +80Da. Then, in the MS/MS fragmentation pattern, pY always stays intact, while pS and pT can stay intact or can lose a neutral loss of 98Da.
How do I use Scaffold for label free quantitative proteomic?
For label free quantitative proteomic, spectral counting is used to get a relative quantification of a protein between different samples. Spectral Count is the sum of all the MS/MS observations for any peptide in a given protein, assuming that the number of MS/MS spectra is related to the protein abundance in the complex mixture of proteins. The criterion that needs to be selected in the Scaffold file is the number of identified spectra.
Which database was used to search my MS/MS data?
If no species is specified, NCBInr will be the default database used for the search. This database is automatically updated every month. For human, mouse, rat or Arabidopsis, IPI databases will be used. If you have ESTs or genomic sequences that can be used for the search (you are working on species not catalogued in other databases), we can create a customized database that will be used for the search.

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2-D Difference Gel Electrophoresis

How much protein is needed for 2-D gel electrophoresis analysis?
In general, 0.5 ml of a sample that has a protein concentration = 4 mg/ml is ideal. For a 7 cm mini 2-D gel, 5 to 25 ug is adequate; for a 24 x 24 cm large gel, 100 to 150 ug of protein load is optimum.
Do you do sample preparation? What sample clean-up procedures do you use?
We prefer liquid protein extract, however we also offer protein extraction services for cultured cells, bacteria, fungi, plant and animal tissue; we also concentrate and process proteins from tears, saliva, serum, plasma, urine, and CSF. We have used different methods such as dialysis, TCA/acetone precipitation, Amersham 2-D clean up kit, ultra centrifugation clarification to remove salt, ionic detergent, nucleic acids, and large particulates etc. The above contaminants can interfere with the first isoelectrofocusing step if they are not removed.
What causes variation in 2-D gel analysis?
First dimension IEF of protein sample is dependant on lysis buffer composition, sample preparation procedure, and total solubilization of the complex protein mixture. SDS concentration, alkylation, and protein entry from IEF strip into the slab gel are important factors affecting second dimension.
Is it necessary to run more than one 2-D DIGE gel for 1 set of samples?
Yes, in order to get the full benefit out of DIGE technology at least triplicates is needed, however four replicates are recommended for a pair of control and treated samples.
Do you perform dye swapping in your DIGE system?
Yes, even though there is no significant chemical variation among dyes we routinely label protein samples with different dyes in same experiment.
How much protein is needed for 2-D DIGE?
For analytical purposes, 50 ug of each sample (control and treated) is needed for each gel for CyDye labeling. Another 50 ug protein mixture made up of equal protein quantity from all samples present in the experiment is labeled with Cy2 as an internal reference, so in all there is 150 ug of total protein loaded in each gel.
Is it necessary to have a pick gel for each DIGE experiment?
It depends, however it is still possible to have protein identified directly from an analytical gel. Higher protein quantity in the spot always increases the chance to get good protein identification results. 1 to 3 mg of a protein mixture made from all the samples present in the experiment including both control and treated can be loaded in a pick gel.
What kind of options do I have for image analysis on 2-D gel data?
There are two options. DeCyder v 6.5 is used for 2-D DIGE gels, this differential analysis software is designed to do automatic spot detection, normalization, background subtraction, spot matching and spot statistical analysis with very little user intervention. For non DIGE gel, we use Progenesis SameSpot software to warp gels, and match spots on different gels, then we use Progenesis PG220 to do spot detection, normalization, background subtraction and statistical analysis; this software is more flexible.
At what instance do I need to use narrow pI range to separate proteins?
It depends. In general, for 2-DGE technique, the spot resolution is very important, if spots appear too close together to be separated confidently then it is better to narrow the pI range.
What is the turn around time of 2-D service?
For a simple 2-D gel, without image analysis, 3 days to 1 week depending on the protein sample. For large gels, such as a 24 x 24 cm DIGE gel, which includes sample preparation, protein quantitation, CyDye labeling, actual 2-D processing, accrued gel images with scanner, spot detection, acquired pick list with differential image analysis, and spot excision, takes almost three weeks.

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Peptide synthesis

How much peptide can you synthesize?
We offer three main synthesis scales: 0.025 mmole, 0.1 mmole, and 0.25 mmole. We are able to purify peptides to 95% by preparative HPLC for an additional cost. Special syntheses are available upon consultation.
What should I expect with my final product?
Each peptide is analyzed by analytical HPLC and MALDI-TOF mass spectrometry before provided to you in freeze-dried form.
How do I store the peptide product?
Keep the peptide dry (in a desiccator) at 4°C. These conditions are best for long-term storage.
I would like to synthesize a peptide based upon protein antigenic site prediction. How do I start and what services do ICBR provide for this purpose?
There are several antigen prediction programs available online. We recommend the links http://bio.dfci.harvard.edu/Tools/antigenic.html or http://www.genscript.com/cgi-bin/tools/antigenic_prediction.pl . After an appropriate sequence is obtained, our lab can synthesize the peptide of interest and conjugate that peptide to a carrier protein (typically KLH). Antibody production services are available through the Hybridoma lab (ICBR Cellomics). We strongly advise consulting with the Hybridoma lab (Linda Green) before starting a project such as this.

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