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> General Procedure |
| General Procedure for Protein
Identification |
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The following is a general process for our Protein Identification
service. The procedure may need to be modified based on specifics of
each project.
- Digestion
A 1D or 2D gel sample is first destained, cleaned,
and digested in-gel with trypsin. Resulted peptides are extracted with
organic solvent, dried down and reconstitute for LC-MS/MS
analysis. A solution sample is first denatured with urea or
Gu.HCl, reduced and alkylated before digestion with trypsin. The digested sample is
then cleaned with reverse phase chromatography to remove urea/Gu.HCl
and other contaminants and then reconstituted in a suitable buffer for
LC-MS/MS. The digestion step typically takes 24 hrs to 36
hours to complete. To achieve the best recovery of peptides, all our
digestions are carried out manually since robotic digestion often lead
to significantly lower peptide recovery.
- LC-MS/MS Sequencing
A Peptide sample from a protein digest is
then injected onto a HPLC with a 75 uM ID reverse phase column. The
HPLC is online coupled with an ion trap mass spectrometer -- the most
sensitive type of mass spectrometer to date. Typically it takes two hours to analyze
one sample by Nano LC-MS/MS.
- Database Search
The MS/MS data, is used to search against the
most current non-redundant protein database from GeneBank, with our
proprietary bioinformatics software to generate a hit list. If requested, we can also search a customer's proprietary protein
database if the proteins of interest are not present in the public
database.
- Validation
The hits proteins are
decided by our mass spectrometric expert based on all experimental
data, bioinformatics analysis and the sample information provided by
our customers. Since most of the hit proteins we reported are based on
two or more high quality peptide ms/ms spectra, our confidence level
is above 99.99%. In the cases that a protein hit is identified by only
one MS/MS spectrum due to very small protein size or very low protein
amount, we will carry out more manual validation by our experienced
specialist (such as manual spectrum assignment) to make sure that each
of such hits is with better than 99% possibility.
- Report
A final report with a list of all the identified proteins and
peptides as well as he description about experimental procedures is delivered to the customer usually within five
business days. In the case that
multiple proteins are identified from one SDS-PAGE gel band we'll
report all the identified proteins to the customer (this is the case
for most 1D gel samples).
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