MassQC
a program by Proteome Software

Bringing Quality To Proteomics

 

 

MassQC Reviewed September 2010

by Andrew Guzzetta, IonSource

Disclaimer Note: We have tried to be as even handed as possible, however we are obligated to disclose that Proteome Software was an advertiser at IonSource at the time this functional review was written.

IonSource.com
 

Program Reviewed: MassQC

Version Reviewed: MassQC Version 1.7

Date Reviewed: September 2010

Vendor: Proteome Software

Web site: http://www.MassQC.com

Use: QC software for LC/MS and proteomics. To date this software only works with the line of Thermo LTQ related mass spectrometers.

Cost: MassQC is server side software with a subscription cost of  $60/month/instrument/academic, and $100/month/corporate.  We have been told that a client side version will be coming soon.

Fully Functional Demo Available at: http://www.MassQC.com

Log In: Create A Free Account

Contact Person for MassQC: marketing@proteomesoftware.com


 

 

 MassQC

review

 

Quality in Proteomics?

The question above piqued our curiosity to review MassQC.  Before jumping into the review we should go over what most people do to assure themselves that their mass spectrometers are working properly.  Most call this test a system suitability test, Proteome Software calls this a QC run.

 Intro

We will go through a QC intro because we know that the concept of a QC run in proteomics will be foreign to some.

Let's assume that you are a mass spectromitrist and you routinely perform protein ID, and more complex proteomic experiments like MudPIT.  Your clients or coworkers trust that you will do a good job and handle their samples with care. From the research side it will often take an extraordinary amount of resources, and research time to come up with a sample for proteomic analysis.  As analysts we understand valuable samples cannot be shot down the drain with a shrug of the shoulders.  Typically we run a QC run before and after the sample set so that we can say our instrument was working before and after the samples ran.  We do this to protect ourselves, and to defend our lab, but more importantly we do it to protect our client's precious samples.

For system suitability, some of us would do a QC run on our LC/MS system using a typtic digest of a standard protein.  Before starting our LC/MS analysis we would inject a digest of a standard protein of known concentration, usually a BSA or a myoglobin digest.  At IonSource we prefer horse myoglobin tryptic digest as our standard, because there is no need to reduce and akylate the cysteines before performing the digest.  As you might be able to tell we like to make our own standard digest.  Also, the myoglobin tryptic digest produces fewer peptides than the BSA digest, which allows us to become more intimate with the peptides that we observe. 

Our QC run at IonSource goes something like this, we inject 10 fmol of myoglobin tryptic digest, and run a short 30 minute gradient, and then look to see if our peptides appear in the base peak chromatogram.  We would also look at the peak intensity as a gauge of our performance and sensitivity.  Then we would submit the QC data file for database searching.  If the search returns with a result for horse myoglobin with 40% sequence coverage, or better, then we say that we passed our system suitability.  We run a post run QC after the sample set, and we pray that we pass.  It is important that we pass our post run QC, because how could we charge our client if we could not say our instrument was still operating after their samples ran.  If you are submitting samples to a CRO (contract research organization) we would suggest that you obtain all of your raw data for archival and request that a QC is run before and after your sample set, request the QC raw files also.

Basically our criteria for passing the system suitability test was protein sequence coverage and peak intensity.  The only real hard, hands off , metric was percent sequence coverage of the correct protein in a database search.  It was not a bad test, and gave us reasonable assurance that everything was working OK.  Our simple test is much better than what most other labs do.  Most other labs virtually do nothing before shooting your precious samples, saying, "Well, it was working, until we got to your stupid samples!  What did you put in there?"  If I were the client I would say, "Prove it!  Prove to me that your stupid instrument was working, or we are not going to pay you!"

 

The Review

MassQC

Program Functional Review

 

 

MassQC evaluates QC runs with a battery of metrics that far exceeds our short glance at system suitability as described above.  

First of all MassQC is a server side application.  We never thought that we would like this software model, but MassQC works seamlessly.  It's nice having a program on a server maintained by professionals.  Version updates are taken care of by Proteome Software, and there is none of this, "Will it work with windows 7?  Do I need to run 64 bit Windows XP?  What do you mean I need to run Linux? What do you mean I need to install .net."  All of that's nonsense is gone, and thank goodness.

MassQC is set up as a subscription based software package. For a low monthly rate you can gain access to the resource.  At $60/mo it is a bargain.  Imagine if you could access Mascot for $60 month and never had to troubleshoot the program, or need to maintain databases.  That would be a dream come true, wouldn't it?  We understand that some private companies, drug companies, don't like sending sensitive data over the internet, and we have been told that a client side version of MassQC is coming soon.  However, we would argue that a myoglobin digest raw file sent over the internet is not a sensitive piece of data.

We will be evaluating the trial version which is the same as the fully licensed version.  The free trial version is good for 30 days.  At the end of the 30 day period we found that we began to rely on it.  The one caveat for the demo is that you need 25 QC runs "in the can" in order for it to start doing its statistical analysis.  Then with each subsequent upload MassQC runs an analysis, and tells you how the most recent run compares with the previous runs. If you only run one QC a day, you will soon be at the end of your trial before you can get to the fun stuff.  When you are doing your demo you can always go back into your log, and pull out old QCs and load them, but it is a bit of a pain to go hunting for them.  We have made some suggestions to Proteome Software to extend their trial period or reduce the number required runs. However, we understand the statistical significance argument as well.  We have some indications that MassQC is addressing this issue, perhaps by allowing the client to see the metrics page before the metrics become entirely significant.

 

If you go to MassQC.com this is what the intro page looks like.

 

When you visit the website click on the "Free Trial" button.  The sign up is pretty nonintrusive, which we liked a lot.  There is not really a marketing component to the signup, and you don't need to give up any private information to get going, which is really cool.  Of course once MassQC sales see this review they will say, "Why aren't we gathering more marketing info?"  The software is very intuitive.  Personally I hate to read instructions and I often go off and just try new programs, and get into trouble, but not with MassQC.  The MassQC interface was very intuitive.  However, when the results come up there are a lot of metrics to take in, and at first it can be confusing, see figure 2 below.   There are 28 metrics, wow!  Actually the documentation for each of the metrics is spelled out in detail.  You will notice a blue question mark at the bottom of each metric.  By clicking on the question mark you will be taken to an in depth description of the metric, see below.  First, if you have a question, click on the question mark, and don't mail Proteome Software or IonSource.  We are guilty of this, mailing Proteome Software asking, "What is this what is that?"  The descriptions provided online are very good.

 

Figure 2.

 

Here we will click on the question mark for peak width, and here is what pops up, see figure 3.

Figure 3.

 

This is only part of the explanation for the peak width query, if you scroll down on the peak width page it tells you how to troubleshoot bad peak shape.  The help section of MassQC is extraordinary.  They have spent a lot of time making every aspect of MassQC crystal clear.

Our first impression of the dashboard, as shown in figure 2, was that we were overwhelmed.  It is a far cry from, "Oh, yeah pretty peaks", and 42% sequence coverage.  We began to ask are there too many metrics, do we need them all?  Do we really need to know the percent of +4 ions?  It reminded us of an airplane cockpit dashboard.  While every gauge may be necessary, it might be nice to filter some of the information.  For example when we get into our cars we want to know how fast we are going, how much gas we have, and is our engine overheating.  If we are doing our weekly maintenance or take the car into a garage we might like to know how much wear is on our brake pads or what our compression is.  So at first it was information overload. Our usual metrics were mixed in with all of these other metrics that we had never considered before.  In short we would like to be able to know everything, but in a functional light we would like to be able to filter.  My guess is that this program will make us all better mass spectromitrists.

There is an option to filter multiple trend lines in the control charts section of MassQC.  Trending is an aspect where MassQC excels.  It is valuable to be able to go back ands see how parameters change over time, see figure 4.

 

Figure 4.

 

The control charts provided by MassQC allow you to see how your instrument is changing over time.  You can definitely see in figure 4 above that our mass calibration has drifted from 1 ppm to 10 ppm over the course of  two weeks; how embarrassing.  Our peak intensity has stayed pretty much the same.  The control charts functionality is definitely a function that we have not had in the past, but it is one that we can see being very valuable.  Before MassQC we would always think, "Our maps looked better in the past than they do now, or are we remembering wrong?"  Things were always better in the past, weren't they?  If we had been running MassQC there would be no doubt, and we would have all the historical data we need.

It looks like we skipped over how you load data, so we will do that now.  It is very simple.  First of all you go to MassQC.com and log onto the account you created.  Then click on the load tab and choose your instrument.  My instrument is called Chara.  See figure 5 below.

 

Figure 5.

Once you are logged in browse to your QC raw file on your local computer, see figure 6 below. It is too simple.

 

Figure 6.

 

Once the new QC raw file is loaded your metrics will be displayed.  The entire loading and analysis process will take only a few minutes.

Figure 6.

Most of the metrics are green.  We are identifying eight peptides in our simple myoglobin QC, which is acceptable to us.  MS2 peaks per spectrum is a bit low but I am not worried too much since we are identifying our peptides of interest.  For this one red metric I might interpret it as I am seeing less noise and more peaks of interest.  I might be tempted to say that this one red metric is useless; however, in another circumstance it might be relevant.  For example if we had two peptide IDs we might look over at the peaks per spectrum metric to see what that number is.  MassQC begins to let you diagnose your instrument related problems in a scientific manner.

 

Conclusion:

MassQC is really the first program that addresses quality control in proteomics.  MassQC addresses an obvious need, just as Scaffold was the first program to address being able to compare multiple proteomic runs side by side. MassQC allows an analyst to evaluate an instruments performance, and whether it is fit to run samples. MassQC also allows a lab manager to monitor the performance of all of  the instruments in the lab from his or her office.  Now a manager can call his assistant to say, "Hey Alice what the heck is going on with LTQ #5, and why the heck did you just load samples?"  At IonSource if we are ever in a position to submit samples to a CRO we will ask if they use MassQC, or we will ask them how they pass their system suitability test, and then we will ask them for their system suitability SOP.  Data quality is also very important in publishing scientific articles.  It would be nice to be able to put a line in the papers methods section like, "Samples were run under these conditions following the run of a QC sample evaluated by the program MassQC passing these metrics."

Program Enhancement or Suggestions: We would like to be able to filter the metrics into two pages, for example a primary metric page, and a secondary metric page.  This way we could reflect our SOP system suitability metrics on the primary page and relegate all of those that may confuse a client to the secondary page.  We would also like to see a base peak representation of the mass chromatogram.  Our dream would be to be able generate a "Passed QC" page that would contain our primary metrics and a representation of the base peak mass chromatogram.  This would be our QC certificate of analysis that we could provide to our clients along with their data to show that we did our part to insure the quality of our analysis. 

We suggest you try MassQC, and check out the free trial.

 

Andrew Guzzetta

IonSource


 

Links:  

  1. NIST: Performance Metrics for Proteomics

  2. How MassQC works

  3. MassQC users forum

  4. Make your own myoglobin digest

  5. Horse apomyoglobin as a system suitability standard.

 

 

 

e-mail the webmaster@ionsource.com  with all inquiries

 

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Last updated:  Wednesday, October 06, 2010 11:23:27 AM

 

 

 

 

 

 

 

 

 

 

 

 

 

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