I am looking for standard proteins that are readily commercially available, not too expensive, are readily digested with trypsin, produce some suitable peptides for LC/MS/MS, and can be used as QC spikes for label-free quantification to see if the analytical system is able to pick up defined concentration differences. Originally I was thinking of coming up with two sets:
Set A which can be used for spiking human/mammalian samples, so peptides should be largely unique in this "background":
Yeast Enolase, Yeast ADH?
Set B which can be used for spiking bacterial samples, so unique against this "background":
BSA, Horse Cytochome C?
Has anyone done this exercise before and can make some recommendations here?
Maybe the iRT from Biognosis is an option for you. They are designed for LC retention time alignment for MRM assays but might also be usefull for your application:
http://www.biognosys.ch/hrm-mrm-tools/irt-kit.html
I understant it that they are fully synthetical, and can easily be spiked into any sample. I know that they are being used routinely and successfully here at the Functional Genomics Center Zurich.
Hello Philip, thank you for the reply.The Biognosys kits are indeed helpful for QC and alignment purposes, however I am additionally looking for mock proteins to perform a differential spike, to demonstrate that e.g. 2x or 5x abundance differences are readily picked up by the assay. This is sometimes a requirement for publishing in proteomics journals, and once you have suitable proteins in hand also very easy to set up.
Hi Christof,
I've often pondered this question myself and the choices are sadly limited for high purity and reasonably cheap proteins. I ended up going for peptides to spike. These clearly don't test for active digestion. I assume you are familiar with the Sigma stds, UPS1 and UPS2? These definitely don't fit the "not too expensive" requirement.
You might want to look at QConCAT (PMID: 21710569). If you have cloning facilities/abilities, it shouldn't be too hard to get a good amount of standard. They can be tailored to requirements. I thought you'd have to DIY, but it turns out this has been commercialised (http://www.polyquant.com/qconcat-technology/). I have no idea on price.
I actually based my spike peptides on these sequences. I varied a few things here and there to ensure a unique sequence.
Good luck,
Peter
Tryptic digest of BSA from Bruker may be a good starting point. I plan to use it for my experiments.
You just need to spike a few proteins from an evolutionarily distant organism. For example, if analyzing human proteomes, you might use a few bacterial proteins at various concentrations. Maybe use a protein from a pathway that uniquely defines the distant organism in order to ensure those sequences don't overlap with your sample. BLAST is useful for this. Also check that the protein you chose will generate a good number of unique peptides after your digest of choice so that you get peptides all across the LC elution gradient. Of course, as stated above you are somewhat limited to cheap, commercially available proteins.
Dear Christof,
we have been looking for inexpensive quantitative standards for method development and QC and I have evaluated a decent number of proteins.
The application and the details have been published in Vasilj, A., Gentzel, M. et al., J Proteome Res, 2012 (http://www.ncbi.nlm.nih.gov/pubmed/22671763). The list of
proteins and further details are in supplement S1. Please note that myosin and actin were not suitable (solubility and multiple isoforms) and were not used.
Best regards,
Marc
Dear Christof,
concerning a commercially available standard: there is a product from Pierce:
Pierce™ Digestion Indicator for Mass Spectrometry, Cat. No. 84841
However, I have no experience with this product and it needs to be spiked-in during digestion (not readily digested as you wished for); nevertheless I just wanted to let you know...
Best wishes,
Alexander
I have recently contributed to the publication of a paper related to this topic: "Benchmarking quantitative label-free LC–MS data processing workflows using a complex spiked proteomic standard dataset" (see link below).
Here is a short description of the standard which was used for this study (taken from the abstract):
"we used a proteomic standard composed of an equimolar mixture of 48 human proteins (Sigma UPS1) spiked at different concentrations into a background of yeast cell lysate to benchmark several label-free quantitative workflows"
The big advantage of using the UPS1 standard from Sigma is the knowledge of the True Positives ground truth. This enables the possibility to calculate False Discovery Rates in the context of label-free LC-MS/MS quantifications (see ROC curves in the paper) and thus to assess the quality of both the LC-MS/MS system and the software used for the quantification.
Peter G Hains said is does not fit the "too expensive requirement", but in my opinion it clearly depends on how you defined the "too expensive" threshold.
Best,
David
http://www.sciencedirect.com/science/article/pii/S187439191530186X
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