Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

The overall goal of this enhanced multiplex method is to increase the number of protein samples that can be analyzed simultaneously while decreasing sample error and instrument time. This method can help answer key questions in the fields of aging, neurodegenerative disease, other age-related diseases, and cancer that are related to pathogenesis, progression, diagnosis, prognosis, and therapeutic targets. The main advantage of this technique is that a comprehensive snapshot of protein changes across many conditions can be generated.

Though this method can provide insight into Alzheimer disease in mouse model, it can also be applied to tissue samples from patient with Alzheimer or a range of other disorders. Generally, individuals new to this method will find it challenging because there are several samples to handle simultaneously in a short time frame. We first had the idea for this method when we recognized that acetylation could be combined with isoberic tagging for protein nitration which motivated us to make the technique work globally for all peptides.

This study will analyze brain, heart, and liver tissues from an Alzheimer's disease mouse model and wild type controls. Transfer 60 to 90 milligrams of each tissue sample to a lysing tube, and add 500 microliters of PBS with eight molar urea. Homogenize the samples using a mechanical homogenizer.

Remove the tissue homogenate from the lysing tube and transfer to a microcentrifuge tube. Rinse the lysing tube with 100 to 500 microliters of PBS with eight molar urea and combine the rinse solution with the tissue homogenate. Centrifuge the tissue homogenate for 15 minutes.

Clutch the supernatant from each sample. After the performing the BCA assay to determine protein concentration, add 100 micrograms of protein from each sample to individually labeled microcentrifuge tubes. Add dithiothreitol to each sample.

And incubate at 37 degrees Celsius for two hours. Add iodoacetamide, IAM, to each sample, and incubate on ice in the dark for two hours. Next, add L-cysteine to each sample, and incubate at room temperature for 30 minutes.

After 30 minutes, add tris buffer with calcium chloride to dilute urea to a final concentration of two molar. Add L1 tosilimitotophenylethylcholer methyl ketone-treated trypsin to each sample. And incubate at 37 degrees Celsius for 24 hours.

On the following day, quench the protein digestion by flash freezing the sample in liquid nitrogen and store at negative 80 degree Celsius until further processing. Prior to dimethylation labeling, the peptide samples are desalted, as described in the text protocol, and dried by vacuum centrifugation. To begin the dimethylation labeling procedure, reconstitute the peptides in 1%acetic acid dissolved in HPLC or nanopure grade water.

Remove a 50 microgram portion of each sample to a new 1.5 milliliter microcentrifuge tube. Store the remainder of the reconstituted peptides at negative 80 degree Celsius for future experiments. In the steps that will be demonstrated next, it is critical to add reagents quickly so the chemical enabling reactions will take place for the same amount of time for all of the samples.

Add eight microliters of a 60 millimolar formaldehyde solution to the samples for labeling with light dimethylation. Add eight microliters of a 60 millimolar carbon 13 deuterium labeled formaldehyde solution to the samples for labeling with heavy dimethylation. Add eight microliters of 24 millimolar sodium cyanoborohydride to the samples labeled with light dimethylation.

Add eight mircoliters of 24 millimolar sodium cyanoboroduderide to the samples labeled with heavy dimethylation. Vortex the samples, and then shake gently on a tube shaker for about 10 minutes at room temperature. Quench the reactions by adding 16 microliters of 1%ammonia for five minutes.

Reacidify the reaction mixtures by adding eight microliters of 5%formic acid. Combine the light and heavy dimethylated peptides to generate a total of six samples. Perform sample desalting as described in the text protocol.

And dry the samples by vacuum centrifugation. To being isobaric tagging of the dimethylated samples, first, reconstitute 100 micrograms of dimethylated peptides and triethylammonium bicarbonate, or TEAB buffer. Next, prepare isobaric reagents according to the manufacturer's protocol of the isobaric tagging kit.

Add the appropriate volume, in this case, 41 microliters of the solubilized isobaric tagging reagent to each of the peptide samples, and vortex for about 10 seconds. Shake the samples on a microcentrifuge tube shaker for about one hour at room temperature. To quench the reactions, add 8 microliters of hydroxylamine to each sample.

And incubate the samples for 15 minutes at room temperature. Pool the six labeled samples into a single mixture and desalt. Prepare the peptides for liquid chromatogrophy by reconstituting the peptides in mass spectrometry grade water with 0.1%formic acid.

Add reconstituted peptides to a microcentrifuge tube containing a 0.65 micrometer filter. And centrifuge at 12, 000 times g for one minute. Remove and discard the filter.

Transfer filtered peptides into an autosampler vial. Inject six microliters of each strong cation exchange faction sample onto a trap column. Pack to two centimeters with carbon 18 material.

Run the analytical separation and data acquisition mass spectrometer methods. A 12-plex cPilot analysis of brain, heart, and liver tissues from an Alzheimer's disease mouse model and wild type controls was performed. Precursor data showed light and heavy dimethylated peptides represented by the peaks at m/z 643.854 and 647.875.

These peptides were selected, independently isolated, and fragmented with collusion induced dissociation to generate these spectra. Search results indicated that the peaks belonged to a peptide of the protein phosphoglycerate kinase one. These peaks were isolated further for higher energy collusional dissociation tandem mass spectrometry and the reporter ions are observed as shown.

Both sets of triple stage mass spectrometry spectra are necessary to get information about the 12 samples. In this example, the Alzheimer's disease to wild type control ion ratios are similar across the two biological replicates for brain, liver, and heart tissues. The fold change values for each comparison suggest that phosphoglyerate kinase one levels in the brain and heart are higher in Alzheimer's disease mice but lower in the liver.

Once mastered, this technique can be done in approximately five hours if it is performed properly. While attempting this procedure, it's important to remember to be careful of sample handling. Incorporated into this procedure, separation methods like strong cation exchange or high pH fractionation can be performed in order to increase per diem depth and coverage.

After watching this video, you should have a good understanding of how to enhance sample multiplexing using C-Pilot.