FAQs
- What size projects do you accept?
- What is the cost for your services?
- How do I get a quote for services?
- How do I ship my sample(s)?
- What is the best method for analyzing my samples?
- How much protein do I need for identification?
- How much protein coverage does a typical identification yield?
- What is the Sensitivity of Protein Identification?
- When is LC/MS/MS analysis required?
- When is de-novo sequencing required?
- Do you need to extract peptides with acetonitrile post-digestion?
What size projects do you
accept?
Any, from single gel plugs to multi-year contracts.
What is the cost for your
services?
You can get per item pricing by contacting us. The final cost is dependent
on the project. The process of working with us starts with a phone conference
to discuss the project and its objectives. The next step is the submission
of a proposal which includes all associated costs.
How do I get a quote for
services?
Please contact Lou Farrell.
How do I ship my sample(s)?
Please follow our sample submission guide. Include a copy of the submission
form with the samples.
What is the best method for
analyzing my samples?
This is very much project dependent. There are multiple ways to compare
samples at the protein level. Many methods can be applied. These range from
just SDS-PAGE to multi-dimensional pre-fractionation followed by 2D gel
electrophoresis. Any method that is chosen will yield slightly different
results in that none of the currently available methods will allow for a
fully comprehensive comparison. In other words, you will not be able to
analyze all proteins that are present in your sample by a single method
since all methods have some bias. We suggest a pilot study that would apply
several techniques and an evaluation of the data in order to determine the
method of choice.
How much protein do I need
for identification?
Although the sensitivity of our mass spectrometers is very high, it is always
prudent to send as much protein as possible. We can identify (or obtain
sequence) from anything that can be seen by coomassie or Sypro'® Ruby
protein stain. Silver is more problematic due to the nature of the stain
and its interaction with the protein but we obtain signal from over 90%
of samples.
How much protein coverage
does a typical identification yield?
Coverage is very much dependent on the amount of protein present and also
the nature of the protein itself. For abundant proteins coverage can be
high as 70-80% whereas low abundance proteins can be identified from as
little as one or two peptides (with sequence information).
What is the Sensitivity of
Protein Identification?
We can identify protein from an SDS-PAGE gel at the 1-3 ng level. Refer
to our technical note addressing sensitivity.
When is LC/MS/MS analysis
required?
We utilize LC/MS/MS for protein identification from 2-D gel spots when MALDI/MS
is not sensitive enough or when there are insufficient peptides to allow
a confident ID by MALDI/MS. LC/MS/MS is also the method of choice for protein
identification from SDS-PAGE bands or to map post-translational modification
sites. Long gradient LC/MS/MS or 2-D-LC/MS/MS experiments are also required
for protein identification from solution digests of complex protein mixtures.
When is de-novo sequencing
required?
De-novo sequencing is required when a species is poorly represented in the
protein or EST databases (or the protein of interest is not yet entered).
It is also useful for protein characterization studies, which may include
mapping post-translational modifications or determining disulphide bridges.
Working with non-tryptic peptides, such as naturally occurring peptides
or those generated from a different proteolytic enzyme (here the fragmentation
pathways under low energy collision-induced dissociation (CID) conditions
can be difficult to rationalize and are not always accounted for by search
engines) also requires de-novo sequencing.
Do you need to extract
peptides with acetonitrile post-digestion?
The majority of protocols for in-gel digestion call for acetonitrile extraction
of peptides from the gel plug in an effort to increase recovery of peptides
(particularly those hydrophobic in nature) from the gel matrix. Subsequent
mass spectrometric analysis requires the removal of organic solvent to facilitate
loading onto reverse phase material (such as ZipTips or LC columns). As
such, peptide solutions are traditionally taken to complete dryness and
reconstituted in aqueous solvent. We have compared this approach with direct
analysis of peptides from the supernatant (in ammonium bicarbonate) without
organic extraction for the analysis of identical gel plugs containing a
known amount of a standard protein. With extensive washing prior to digestion
and careful control of volumes during the digestion itself, the analysis
of digest supernatants directly yields sufficient peptide amounts for protein
identification (see ‘In-Gel Digestion’ technical note).