FAQs for MALDI-ToF/ToF and LC/MS/MS Services

Q1) How much material do I need to prepare for protein identification by MALDI-TOF/TOF?
Ans: As long as the protein is visible in a Coomassie stain of your gel separation, there is sufficient material to generate a good analysis. By providing us with more than 200 fmol (or 10 ng of a 50 kDa protein) you ensure a maximum probability of success. If you are providing your sample as a solution or a lyophilized fraction, try your best to provide at least 100 ng for each spot (the more the better).

 

Q2) Can I use silver staining on a sample prepared for MS?
Ans: In order for silver stain samples to have a chance of working on the MALDI instrument, you need to use the altered silver stain method for MS.

Silver staining Protocol compatible with mass spectrometry analysis

  1. Allow at least 250 ml of each solution per gel.
  2. All steps are carried out at room temperature with gentle shaking.
  3. Make all solutions freshly when needed. Make sure to use only double distilled (18.2 MW) water. All solutions should appear clear and colourless before they are poured onto the gel.
  4. Assuming that the gel has been run attached to a backing material, it should be fixed in a solution of 30% ethanol/7.5% acetic acid for at least 2 hours.
  5. Sensitize gel in the sensitizing solution for 30 min
Reagent Quantity
Ethanol 75 ml
5% Sodium thiosulphate solution 10 ml
Sodium acetate 17 g
ddH20 To a final volume of 250 ml

Table 1. Sensitizing solution. 

  1. Rinse the gel three times in ddH2O, for five minutes each rinse.
  2. Incubate in a 0.25% silver nitrate solution for 20 minutes.
Reagent Quantity
Silver nitrate 625 mg
ddH20 To a final volume of 250 ml

Table 2: 0.25% Silver nitrate solution. 

  1. Rinse the gel twice in ddH2O, for five minutes each rinse.
  2. Incubate the gel in developing solution for up to 10 minutes. If the staining is intense enough before the end of 10 minutes, move directly to the stop solution.
Reagent Quantity
Sodium carbonate 6.25 g
Formaldehyde 100 ul
ddH20 To a final volume of 250 ml

Table 3: Developing solution. 

  1. Pour the developing solution off and add the stop solution, incubate for 10 minutes.
Reagent Quantity
EDTA 3.65 g
ddH20 To a final volume of 250 ml

Table 4: Stop solution. 

  1. Rinse the gel three times in ddH2O, for five minutes each rinse.
  2. Store the gel in ddH2O.

 

Q3) How to send the sample in whole gel form (without excision)?
Ans: Gel image of your protein sample need to be included. Unused gel bands will not be return back. There is no extra charge for the excision in this case. Put the gel in a sealed, airtight bag, with minimal volume of Milli Q water. Gel can be rolled and put in a tube or put between two flat pieces of cardboard. Wrap in anti-crush (bubble wrap) material. The quarantine letter should be attached next to the gel: This package contains only non-hazard proteins separated by protein gel electrophoresis.

 

Q4) How do I avoid keratin contamination?
Ans: Best results are always obtained from clean samples. Contamination occurs primarily from keratin proteins from hair and skin. To avoid keratin contamination, the use of gloves when handling samples is recommended.

 

Q5) Is pooling required for silver or Sypro stained gels?
Ans: Yes.  For silver we recommend pooling of two spots; and 4 spots for Sypro ruby or Cy dyes. For low abundance samples of this type of staining, we would use high sensitivity nano-LC MS/MS instrument.

 

Q6) What is the treatment required for Sypro ruby stained, silver stained or Coomassive stained gels respectively?
Ans: All proteins will be de-stained prior to analysis. No pre-treatment is required. However, the type of stain used is important:

  1. For Coomassive there are no extra requirements.
  2. For silver stain, a mass spectrometry compatible procedure must be used.  Any gels stained with a traditional silver stain method that uses gluteraldehyde cannot be analyzed by mass spectrometry.
  3. Several suppliers offer silver stain kits that are mass spec compatible (e.g. Amersham International: Plus-One Staining Kit).

 

Q7) I have an old sample, can it still be analyzed?
Ans: Silver stained gels older than 1-2 months may not yield satisfactory results.  Coomassive stained spots and bands can be analyzed even if they are several months old.

 

Q8) Can samples be analyzed in liquid form?
Ans: Yes providing the sample is pure and in a low salt buffer e.g. 50mM ammonium hydrogen carbonate; for TRIS or phosphate buffers the maximum salt strength is 20mM. NaCl and surfactants (e.g. SDS) cannot be present. The sample must be freeze dried or lyophilised in a micro-centrifuge tube prior to shipping. NOTE: The type of buffer and the volume of the liquid before drying must be stated on the Sample Form. Complex liquid samples containing several proteins can also be analyzed in some circumstances. Please consult us before submitting samples in this case.

 

Q9) Can other enzymes (beside trypsin) be used to digest the protein?
Ans: All standard analyses use trypsin digestion because it is the most efficient enzyme for the proteomics pipeline, however, other enzymes such as chymotrypsin, and gluC can be used if required. 
This must be clearly indicated on the Sample Form. 
** Additional charges apply, please enquire.

 

Q10) Can the proteins be sent after enzyme digest?
Ans: No. Since PI is an accredited facility all samples must be processed alongside a Quality Control sample, and this is not possible if the sample has already been digested.
 

Q11) Is the protein reduced and alkylated prior to analysis?
Ans: Reduction and alkylation of cysteine is not performed in Service SS001 because it is not required to achieve protein identification. Sometimes cysteine containing peptides are not detected but the numbers of peptides affected is small compared to the total sequence. NOTE: It is recommended to reduce and alkylate low mass proteins (<20kD) before they are run on 1D or 2D gels.
 

Q12) Can I analyse a low mass protein?
Ans: For proteins with a low mass (<20kD) it can be difficult to obtain a significant protein identification. In this case larger amounts of sample will be beneficial. Electrospray (LC/MS/MS) mass spectrometry will be required