Complex Mixture ID

Introduction

Modern LC-MS/MS platforms can identify hundreds or even thousands of proteins from a single sample. Our instrumentation allows the characterization of complex samples without the need to resolve on a gel and excise bands. Comparison between complex samples is often better performed by complex mixture analysis than by running a gel and cutting out visibly different bands. The mass-spectrometer will observe more differences than are visible with Coomassie or silver staining.

We offer complex mixture analysis from gel – you run your sample into the top of the resolving portion of a gel, cut this region out and deliver it to us with staining. Very complex samples can be run further into the gel and cut into multiple slices (see below). IT IS CRUCIAL NOT TO INCLUDE ANY OF THE DYE FRONT OR STACKING REGION OF THE GEL WHEN CUTTING AS THESE CAN SIGNIFICANTLY AFFECT THE RESULTS!

What Does it Cost?

$180 per 5mm slice of gel. Each sample submitted must consist of no more than 10mm of a gel lane. Very complex samples can be run into the gel more than 5mm, cut and submitted as multiple slices for improved coverage. See charges page for details of discount for large number of slices from single lane or condition.

What We Do

We run your samples on our Orbitrap Elite or Orbitrap Fusion Lumos mass spectrometers, using a short reverse-phase LC-MS/MS method. We identify proteins from samples using Proteome Discoverer 2.2 and e-mail an Excel summary of identifications. We can provide a single report comparing the content of multiple samples, including label-free quantitation (at no extra charge).

What You Must Do

To submit a complex mixture sample you must:

  • Run your sample 5-10mm into the resolving part of a pre-cast SDS PAGE gel. Your sample should proceed into the resolving area, and not remain in any stacking area of the gel.
  • Stain your gel with Coomassie Blue – all complex mixtures must be stained.
  • Cut out the stained area as one or more 5-10mm slices. Each slice will be a separate sample, analyzed and charged. Do not cut out the very top of the gel (loading/stacking) area, which is likely to have detergent from your buffer on it’s surface.
  • Adhere to our guidance when cutting out the band, ensuring you dice each band into 1mm cubes (no smaller) etc. and ensure your slice(s) are no larger than 10mm of gel.
  • Place each slice into an Eppendorf 1.5ml tube, which has been rinsed with 50% organic solvent and millipure water.
  • Follow the general guidelines for sample submission.
Comparative / Quantitative Analyses

We offer free label-free quantitative comparison for complex mixtures. It is useful to identify larger differences in abundance of proteins between samples. Accuracy depends on the complexity of the sample, and abundance of the proteins quantified. It is not suitable to accurately quantify relatively small fold-changes, e.g. 2-fold or less (dependent on sample).

Contact us before submission if you would like a quantitative analysis. Depending on your experiment it is likely that biological and technical replicates will be required for a useful outcome.

Depth of Coverage

The Orbitrap Fusion Lumos instrument currently used for most service work can identify up to approximately 4000 proteins per run from an abundant whole lysate. However, depth of coverage depends on the the amount of protein present, and not exceeding the maximum capability of the mass-spectrometer. If your sample is likely to contain >3000 proteins you should consider running it into the gel further than 5mm, and cut out multiple 5mm bands to be run on the instrumentation separately. If you require high sequence coverage of identified proteins you will need to ensure your sample contains enough material, and cut the gel into multiple slices containing <3000 proteins per slice. Contact us if you have any doubts. Running a silver-stain gel using a small amount of the sample can be useful to decide the correct number of slices that should be cut for the complex mixture analysis, as it allows us to visualize the complexity of the sample prior to performing the actual analysis. We can also perform high-pH fractionation on your sample.

Warnings & Limitations

Complex mixture analysis on our instrumentation is powerful and very cost-effective. However, it is important to match the complexity of your sample to an appropriate run-time on the mass-spectrometer by cutting out an appropriate number of gel slices. Consider speaking to us before submitting your first complex sample, or if you have any doubts about the complexity of your sample.

We can run searches for post-translational modifications against complex mixtures, but are not able to review and validate results due to constraints on staff time – this is left to the customer. We provide a more comprehensive PTM service for gel bands containing purified protein.

Any Questions?

If you have any questions please contact ProteomicsCore@UTSouthwestern.edu before you prepare and submit your samples.

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