Frequently Asked Questions

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General

Q. How can I get good MS results?

Q. How should I store my proteins?

Q. How can I avoid keratin contaminants?


Sample Preparation

Q. Why is sample clean up necessary?

Q. When is sample cleanup needed?

Q. What is ZipTip used for?


Data Analysis

Q. How do I know if the database search results are "good matches"?

Q. What are Ions Scores and Protein Scores?

Q. What is Confidence Interval (C.I. %)? How do I use that to interpret my results?

Q. Besides database searching scores, what other independent evidences are there to support the protein identity?


Money Matters

Q. What is the definition of "PI" and who is eligible to be one?

Q. Is there any volume discounts (for larger number of samples)?

Q. When will I receive the invoice for the service performed at CPOS?

Q. Can I pay in advance for services that will be performed later?

Q. Can non-HKU academic institution or commercial (for-profit) organization use CPOS's services? What will be the rate?



Q. How can I get good MS results?

  1. High purity sample is the key to good MS results.
    Minimize contamination as much as you can – keratins, polymers, Sephadex / Agarose beads, detergents, and non-volatile buffers can adversely affect sample signal and they are very difficult to remove!
    Use HPLC to purify samples whenever possible. Meanwhile, 1D gel band or 2D spot band containing predominantly one protein will also give satisfactory data. Analysis of complex protein mixtures may not be feasible and not likely to give good results due to lowered protein score and ion suppression effects.
  2. Sufficient sample quantity for protein identification.
    In most cases, a minimum concentration of pmoles/uL is required. Higher concentration is needed for high MW protein and when high sequence coverage is desired (such as Post-Translational Modification (PTM) analysis). Please refer to Sample preparation guidelines.
    For gel plugs, if sample spots can be observed by Coomassie Blue staining (detection limit 100 ng) then there should be enough for protein identification.
  3. Minimize salt concentration in samples.
    Avoid phosphate buffer, detergents, salt and ammonium salts or derivatives of organic amines.
  4. Minimize the number of steps required for purification to minimize peptides loss.
  5. Use low-binding (not silanized) tips/tubes/plates.
    This is to minimize protein/peptide loss by adsorption of the peptides to the plastic. Extraneous peaks caused by plasticizer and other contaminants has been found shedding from lower-quality plastics and interfering with downstream MS. Brands that have worked well in our lab: Eppendorf, Sorenson, Avant, ART.
  6. Keep sample volume small and minimize contact surface area.
  7. Avoid complete drying of protein samples.
  8. Avoid repeated freeze and thaw of samples.

Q. How should I store my proteins?

Proteins in gels plugs are relatively stable at room temperature or 4°C. Store the gel plugs in clean microfuge tubes with 1.0% Acetic Acid to prevent microbial growth.

Proteins in solution may not be stable and could be susceptible to proteases degradation and MUST be kept frozen at -20°C or -80°C. Please submit your samples for analysis as soon as possible after they are isolated and purified.


Q. How can I avoid keratin contaminants?

  1. Wear gloves and long sleeve lab coat when preparing samples.
  2. If possible, work under laminar flowhood to minimize contaminations.
  3. Use only high quality water e.g. MilliQ water in all steps.
  4. Keep workbench free from dust. Hair, dead skin cells, wool sweaters, saliva and dust are common sources of keratin.
  5. Wipe all equipments with ethanol before handling samples: pipette, speedvac, etc.
  6. Keep gel boxes and staining boxes clean. Dirty gel staining boxes have been cited as a major source of keratin contamination.
  7. Never touch the gel with bare hands.
  8. Keep gel boxes and gel containers closed.
  9. Prepare fresh buffers and solvents regularly.

Q. Why is sample clean up necessary?

Contaminations can adversely affect matrix crystallization, mass spectrometry signal and sensitivity, which in turn impact the ability to identify peptides and yield lower sequence coverage.

Whenever possible, avoid using Triton, Tween, SDS, or PEG during any step of protein preparation. Even a trace amount is very difficult to remove.


Q. When is sample clean up needed?

Sample clean-up is needed when proteins...

  1. are prepared in phosphate buffer.
  2. have ammonium salts or derivatives of organic amines (ammonium bicarbonate, Tris HCl) in concentrations greater than 10 mM.
  3. are contaminated with detergents or polymers.

For i and ii, we recommend using ZipTipC18 to cleanup peptides, proteins and oligonucleotides.

For iii, Reverse-phase HPLC purification can most effectively cleanup sample and should be used whenever possible. Microcon centrifugal filter device (Millipore) is effective in removing small molecule impurities, small polymers and certain detergent contaminants with its size exclusion membrane but not so effective with removing others contaminants (such as Tween and Triton) from small protein. Please refer to the manufacturer technical guidelines (Millipore) for more details.


Q. What is ZipTip used for?

ZipTipC18 (or C4) (Millipore) is used for desalting and concentrating of peptides and proteins from aqueous solutions. It is also effective in removing small-molecule contaminants. (fig.1b,c)

On the other hand, do NOT use ZipTipC18 to remove detergent as they will bind with the reverse-phase material and concentrate with the peptides! (fig.1d)

Finally, please note that the typical recovery rate of ZipTipC18 clean up ranged from 50%-60%. (fig. 1a)

Please refer to the manufacturer technical guidelines (Millipore) for more details.


Fig. 1 - Effect of various common contaminants on digested Beta-Gal MS signal.
(A) Digested Beta-Gal Control before and after ZipTip clean up.
(B) and (C) Sample signals suppression from the addition of 0.05M DTT and 0.5M NaCl respectively. Signal intensity improved several folds after ZipTip clean up.
(D) Suppression of sample signal with the addition of 0.5% Tween20 before and after ZipTip clean up.

Q. How do I know if the database search results are "good matches"?

Mascot employs probability based scoring to provide a statistical basis for scores and also to estimate the probabilities of random matches. Protein Identification using statistical approach allows users to evaluate their results by reviewing the Protein/Ion score and by Confidence Interval (C.I.)%.

For each database search, the Mascot search engine calculates a Significance Level. Protein Scores (MS data) and Ion Scores (MS/MS data) above the Significance Level are considered to be statistically non-random at the C.I. 95%. You can compare Protein Scores or Ion Scores to their respective Mascot Significance Level to evaluate how good the results are.

Alternatively, you can compare the C.I.% from different database searches. The closer the Confidence Interval (C.I.%) is to 100%, the more likely the protein is correctly identified.

Please refer to the FAQ Questions What are Ions Scores and Protein Scores?” and "What is Confidence Interval (C.I.%)? How do I use that to interpret my results?" for more information.


Q. What are Ions Scores and Protein Scores?

Protein Score – in a [PMF] analysis, the protein score is calculated by the Mascot search engine for each protein matched from the MS peak list. This score is calculated on P, the probability that peptide mass matches are non-random events. Protein Score = -10Log P.

If the Protein Score is equal to or greater than the Mascot Significance Level calculated for the search, the protein match is considered to be statistically non-random at the 95% confidence interval.

Ion Score – in a [MS/MS] or [PMF+MS/MS] analysis, the ion score is calculated by the Mascot search engine for each peptide matched from MS/MS peak lists. This score is based on the probability that ion fragmentation matches are non-random events. If the Ion Score is equal to or greater than the Mascot Significance Level calculated for the search, the protein match is considered to be statistically non-random at the 95% confidence interval.

Example: In each of the MS Report you receive, you will have a Protein Score for each of your query. Let’s say if the protein score is 150 and the significance level is 67, there is less than a 1 in 20 chance that the observed match is a random event. Similarly, ion scores can be interpreted the same way in which it needs to be above the significance level to be statistically non-random.

IMPORTANT: In a [PMF+MS/MS] analysis (which is the default database search analysis at CPOS), the Protein Score is a combination of PMF-type Protein Score and Total Ion Score.

I.e. Protein Score [PMF+MS/MS] = Protein Score [PMF] + Total Ion Score [MS/MS].

Total Ion Score – in a [MS/MS] or [PMF+MS/MS], the total ion score is calculated by weighting Ion Scores for all individual peptides matched to the protein that is associated with this peptide and MS/MS spectrum. Ion scores for duplicated matches are excluded in the calculation.
I.e. Total Ion Score for Protein A = ion score 1 for Protein A + Ion score 2 for Protein A…etc

Note that this score is NOT statistically rigorous. A bunch of low-scoring (and therefore, statistically insignificant) peptides can match a given protein and contribute a high Total Ion Score.

For more information on search results and Mascot scoring, please refer to Mascot Online Help. (www.matrix-science.com)

Summary

  Where can you find this Note
 Statistically significant
 Ion Score
In a [MS/MS] or [PMF+MS/MS] analysis
Statistically non-random at the 95% confidence interval if above MascotR Significance Level
 Protein Score*
In a [PMF] analysis
Statistically non-random at the 95% confidence interval if above MascotR Significance Level
 Not statistically significant
 Total Ion Score
In a [MS/MS] or [PMF+MS/MS] analysis
Low-scoring (and therefore, statistically insignificant) peptides can match a given protein and contribute to the Total Ion Score.
 Protein Score*
In a [PMF+MS/MS] analysis
Protein Score is a combination of PMF-type Protein Score and Total Ion Score.

*In a [PMF+MS/MS] analysis (which is the default database search analysis at CPOS), the Protein Score is a combination of PMF-type Protein Score and Total Ion Score.


Q. What is Confidence Interval (C.I.%)? How do I use that to interpret my results?

Comparing Protein Score or Ion Score from different searches is not straight forward because different database searches have different Mascot Significance Levels (which depends on the size of the searched database and on the numbers of masses submitted for a database search). The C.I.% is a statistical calculation based on normal probability distribution that allows you to directly compare the results from different searches.

C.I.% rates the confidence level of the Protein Score or Ion Score. If the score returned from the search is equal to the Mascot Significance Level for the search, the score is given a 95% Confidence Interval. Scores that are higher or lower than the Mascot Significance Level for the search are given higher or lower Confidence Intervals, respectively. The closer the Confidence Interval (C.I.%) is to 100%, the more likely the protein is correctly identified.

C.I.% results are given for the following scores:

  • Protein Score C.I. % [MS]
  • Ion Score C.I. % [MS/MS]
  • Total Ion Score C.I. % [MS/MS]
  • Best Ion Score C.I. % [MS/MS]

Summary

If Protein or Ion score > Mascot Significance Level,
then Protein or Ion C.I.% > 95%

If Protein or Ion score < Mascot Significance Level,
then Protein or Ion C.I.% < 95%

*Note: If the calculated Confidence Interval (C.I. %) is >100, then 100 is reported. If the calculated Confidence Interval (C.I. %) is < 0, then 0 is reported.


Q. Besides database searching scores, what other independent evidences are there to support the protein identity?

  • Look for protein result that matches proteins from the expected species or kingdom. Mascot search is restricted to a particular species using the taxonomy filter if specified.
  • Show consistency between experimental size and pI of the protein in 2D gel separation and the size of the top scoring protein search result.

However, the following points should be considered:

Many sequence databases do not provide species information in a systematic and rigorous form. Depending on the protein, it may be removed by the taxonomy filter.

  • Contaminants can never be ruled out, and could come from any species, e.g. BSA or keratins. Refer to our tryptic peptide exclusion list here.
  • Unless the genome of the species of interest is completely sequenced, there is no guarantee that the true sequence of the analyte protein is actually present in the database. If it is missing, then high scoring matches from other species are of interest because they are likely to be homologous to the unknown.

Q. What is the definition of "PI" and who is eligible to be one?

PI stands for Principle Investigator. Most PI's are academic staffs who hold a funding account. In short, PI is whoever will eventually pay for the services.


Q. Is there any volume discount for larger number of samples?

We do offer volume discounts for large orders. Please refer to Service Charges & Ordering for more information.


Q. When will I receive the invoice for the service performed at CPOS?

CPOS issues invoices monthly at the beginning of each month for services completed within the previous month.


Q. Can I pay in advance for service that will be performed later?

Under normal circumstances, we do NOT take advance payment for local HK clients. Advance payment is required for non-HK clients AFTER job is confirmed.


Q. Can non-HKU academic institution or commercial (for-profit) organization use CPOS's services? What will be the rate?

Yes. We welcome non-HKU clients to use our service as well. Charges to external users are priced to include Facilities and Administrative costs.

Please refer to Service Charges & Ordering for more information.


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