Proteomics Laboratory | F.A.Q.

1. How much protein do I need?
Any band that can be detected with Coomassie staining contains enough protein for a successful analysis. In my experience, this requires approximately 1 pmol of protein, but the exact amount is dependent on the protein. The protein must be visualized to be cut from the gel.

2. What about analysis of silver stained bands?
The analysis of silver stained protein bands is certainly possible with mass spectrometric methods. The mass spectrometry is amply sensitive for the analysis of fmol amounts of protein so it is fundamentally able to sequence silver stained bands. However, two significant practical problems must be addressed and can destroy the effectiveness of the experiment. 1) Glutaraldehyde fixation modifies proteins in a manner that makes them impossible to digest. This reagent simply cannot be used in the staining procedure. 2) The background created by both protein and non-protein contaminants of the gel will mask the signals produced by peptides derived from the protein of interest. These contaminants are certainly also in Coomassie stained gels, but the inherently larger amount of analyte protein overcome the problems these species create. As a result, the ultimate success of experiments sequencing silver stained bands is generally dependent on absolute cleanliness and care at every stage of the experiment, including the sample preparation and electrophoresis step. It has been my experience that most laboratories find that it is easier and quicker to scale up to Coomassie blue-detectable amounts of protein than it is to identify and solve contamination problems.

3. What is the turnaround time?
Our goal is to be able to provide preliminary data within two weeks and a written report within three weeks.

4. How pure does the sample need to be?
Any protein that can be cut from a gel can be sequenced, no matter how many additional bands are present in the gel. There will always be some concern that a band is composed of more than one protein, but this should not affect the ability to detect and sequence peptides produced in the digestion.

5. Can I identify post-translational modifications?
Post-translational modifications can be identified by mass spectrometry; however these experiments generally require more protein than the identification experiments. This is especially true for modifications that are sub-stoichiometric in nature. One of the inherent difficulties in these types of analysis is identifying a low abundant modified peptide present in a complex peptide mixture. Several different strategies can be sued to increase the likelihood of success in these experiments including large amounts of protein, increasing the stoichiometry of the modification reactions, using targeted analysis and multiple proteases.

6. Can the amounts of my protein or post-translational modification be quantified?
Relative quantities of proteins and post-translational modifications can be determined in these experiments. In order to achieve this, an internal standard must be utilized as a normalization factor. Fortunately, in most samples an unchanging tryptic peptide inherent in the sample can serve as an internal standard.