Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication

The overall goal of this method is to use multiple reaction monitoring mass spectrometry to identify and quantify the species present in raw meat mixtures for use as a tool for food fraud detection. This method can help answer key questions in the field of food integrity, such as confirming the species present in products like sausages or burgers. Our approach is based on mass spectrometry of the protein component.

The main advantage of this technique is that it can give a rapid and quantitative outcome. We first had the idea for this method when we noticed the proteins known by the same name and different species are actually different enough to give species identification, but similar enough to allow quantitation. Thermally denature purified reference myoglobins by heating the sample in a hot block at 95 degrees celsius for 30 minutes.

Cool the sample for approximately 15 minutes until it reaches room temperature. Then add 30 milligrams of urea to enhance the digestion and mix. Add a trypsin solution to the sample in a one to 30 ratio of enzyme to substrate by weight.

Then mix by gentle vortexing and allow it to proteolyze overnight at 37 degrees celsius. Next desalt the post proteolysis sample by first diluting the sample one to two volume to volume with water. Activate a polymeric reversed phase cartridge filled with 30 milligrams of reverse phased material by adding one milliliter of methanol.

Then equilibriate the cartridge by adding one milliliter of one percent formic acid. Load the sample onto the cartridge under gravity. Now wash with one milliliter of five percent methanol containing one percent formic acid.

Elute the peptides with one milliliter of acetonitrile water into two milliliter microcentrofuge tubes prefilled with five microliters of DMSO. Next remove the solvent under vacuum at 50 degrees celsius using a centrifugal evaporator for 120 minutes. Then redissolve the residue in 250 microliters of acetonitrile water.

Transfer the solution to a low volume autosampler vial. Locate the myoglobin sequences for the different meets in the UniProt database. Enter the myoglobin sequences into the target box of the peptide and transition prediction software.

If required, hover over a peptide to reveal its fragment list. After inputting preferences as described in the text protocol, click on Export and select Transition List to create a spreadsheet containing the generated MRM transitions and parameters. Set up a high performance liquid chromotography or HPLC system of a binary gradient with an autosampler and a C18 core shell HPLC column connected to a triple quadrupole mass spectrometer operated in positive electrospray mode with MRM detection.

In the edit parameters section of the liquid chromotography mass spectometry data collection software, select the scan type as MRM and the polarity as positive. Input the Q1, Q3, time, ID, DP, and CE values for the transitions created in the spreadsheet and save the acquisition method. After setting up the method parameters as described in the text protocol, navigate to the data collection software, click on Acquire and select Equilibrate.

In the box that opens, select the required acquisition method to begin the instrument equlibration. Next put the sample vials in a rack in the autosampler. Click on File and select New, followed by Acquisition Batch.

In sample name insert the identity of each of the samples to be analyzed. See the text protocol for details on how to visualize the acquired data. Use a meat that has been previously frozen and then ground into a powder.

Prepare a range of meat mixtures by weighing respective amounts of meat into 15 milliliter plastic centrifuge tubes. Add four milliliters of extraction buffer to the meat powder. After vortexing for 30 seconds, extract on a lab shaker at room temperature for two hours at 250 cycles per minute.

Transfer two milliliters of the extract into a two milliliter microcentrifuge tube and centrifuge for five minutes at four degrees celsius and 17, 000 G.Transfer 200 microliter aliquots of the supernatant into two milliliter centrifuge tubes. Dry the samples using a centrifugal evaporator before determining the protein concentration of the samples as described in the text protocol. To perform proteolysis of meat mixtures, redissolve the dried residue in one milliliter of 25 millimolar ammonium bicarbonate solution.

Mix well by vortexing and perform proteoloysis as before. Then set up and run the LCMS in a similar manner using scheduled MRM. Display the full chromatogram in the data viewing software.

Display the extracted ions for each transition set in turn. Visually confirm each cluster contains the requisite number of bell shaped peaks at the expected retention time. Thereby confirming the existence of the selected peptide.

Next double click Quantiation Wizard in the navigation bar. In the Select Samples window, create quantitation set by selecting a single data file. Then select one or more available samples.

Select Next to display select settings and query box. Leave the default settings and select Next to display Select Method. From the dropdown Method box, select the integration method file, finally select Finish.

Save the results table, export as a text file, and open it in a spreadsheet to review the data as described in the text protocol. This figure shows the most intense MRM transitions for horse obtained from a one percent weight for weight mixture with beef. In contrast, the red line shows the transition from beef when there is no horse present.

This is a plot of the measured versus the prepared amount of horse in beef expressed as ratios. The plot clearly shows how the method can be used for making quantitative measurements. Once mastered and when using batch processing, the throughput by this method can reach up to 20 samples per 24 hours.

While attempting this procedure, it's important to remember to select a suitable protein for extraction analysis. Myoglobin is a good candidate for examining red meat species because it is plentiful, water soluble, and heat stable. This procedure can be extended to include further species to be identified and quantified simultaneously.

The maximum number is limited only by the mass spectometry scan speed. This technique is paving the way for researchers to use pepto fingerprinting in areas such as consumer and brand protection.