Frequently Asked Questions
- What kind of service does protein identification include?
- What is the turn-around time?
- What happens if we send CFG a sample for analysis and for whatever reason, CFG could not generate any data?
- How much protein coverage does a typical identification yield?
- How much material do I need for the protein analysis?
- Could I do protein ID from the western detectable bands?
Q: What kind of service does protein identification include?
A: We provide a protein identification using Q-Tof 2 LC-/MS/MS. This includes in-gel digestion, LC-MS/MS analysis, database searching, and report generation (electronic versions).
Q: What is the turn-around time?
A: The typical turn around time is around 3- 5 days for protein identification from gels. For 2D-LC-MS/MS analysis and 2D gel electrophoresis, it takes about one week to complete the whole procedure.
Q: How much protein coverage does a typical identification yield?
A: 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).
Q: How much material do I need for the protein analysis?
A: 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 SyproÔ 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. Listed below are some arbitrary numbers for you to get started.
|Theoretical required total starting protein amounts for individualprotein visualization by staining|
|Protein abundance, copies per cell||Silver staining*||Coomassie staining*|
|Protein amount, mg#||Number of cells||Protein amount, mg#||Number of cells|
* Protein detection limits for silver and Coomassie staining were 1 and 100ng respectively.
# Soluble yeast protein was calculated based on 1 mg of yeast protein being derived from harvesting 6x107 cells. Calculations are based on a protein molecular mass of 50 kDa and 100% efficiencies of the procedures used.
* Gygi, SP, Corthals, GL, et al (2000), PNAS, 97(17), 9394
Q: Could I do protein ID from the western detectable bands?
A: This is always a gap between mass spectrometry and western detectable range. Antibodies used in Western blots can be incredibly sensitive. A good monoclonal antibody can pick up low pg of antigen. If the detection is by ECL, the sensitivity is relative to the signal to noise. We often see blots of phosphotyrosine containing proteins blotted with the phosphotyrosine-20 antibody and yet a stained gel shows no sign of protein band, phosph or not! These are often levels way below what is usable for a protein ID by mass spectrometry. To get an idea of the amount of protein you are dealing with, you could get hold of some of the recombinant antigen and run some semi-quantitative westerns. If your sample signal is equivalent to sub-ng amounts, then protein ID by peptide mass fingerprint with or without ms/ms is likely to be unsuccessful.