Ok, this is a great question (thanks for asking!). We have a number of quality control procedures we do before and during the analysis of your sample. Every facility will do this a bit differently, but this is how we do it. It is also evolving as new technology and software becomes available.
We practice bottom up proteomics, which means that your protein sample is digested into peptides before we analyze them by LC-MS/MS. This digestion step is crucial and if it fails, nothing will work. When we digest your sample, we also digest a known control sample. Usually this is a gel piece from a gel of a human cell lysate. If your sample looks unusual or shows signs of a failed digestion (rare, but it does happen), excess keratin contamination or polymer contamination we analyze the control digestion and compare it to our historical data of that control sample. If the control digestion has failed we will contact you and apologise profusely. We will also of course not charge you. This type of failure does happen, but it is rare.
Nano-Scale LC-MS/MS is tricky and the sensitivity and chromatography of our machines can vary (sometimes dramatically). So how do we control for this and how do we know when our machines are performing well enough to run your samples. We have two types of controls we use for this step. The first is replicate control runs of 125 fmol of a BSA digest. We buy this BSA digest from Michrom Bio-resources and analyze them right before we run your sample. Here is an example of a recent control run (July 2011). We then make 7 extracted ion chromatograms for 7 peptides from this digest that span the entire gradient. We look for such things as peak shape, intensity, retention time, etc..
We then run the same sample again and look for retention time reproducibility.
Finally we search the BSA QC run and look for the number of peptides identified, sequence coverage etc… We store all this data in an excel spreadsheet and currently have historical data going back several years for every LC-MS/MS system we own. So we know before we run your sample, how the machine is behaving and how it’s performance compares to past performance.
We also have a more complex QC (Human cell lysate) which we run every few weeks or when we have have a suspicion something is wrong. We do not run this sample daily because running such complex samples usually will degrade the machines performance after the run is complete, unlike our BSA control.
If you have any other questions please give us a call or send us an e-mail.
Last updated on July 14, 2011 by Brett S. Phinney, Ph D.