The overall goal of this procedure is to compare the concentrations of many proteins between samples by mass spectrometry using ready proteomics. This is accomplished by first digesting the protein from each sample to generate peptide mixtures. Next, the two samples are mixed, and peptides in the mixed sample are fractionated and desalted.
Next, the mixed sample is then analyzed by mass spectrometry. Finally, the peptides are identified by comparing the MS two spectra to a target decoy database of all potential peptides in the samples and the relative abundance of peptides between the samples are quantified. Ultimately, ready proteomics enables the quantification of the relative abundance of many proteins between complex samples.
The main advantages of reductive demethylation over other methods for stable isotope labeling such as iLite, is that ready is a fast, inexpensive, and accurate method that does not require specific trois or a synthetic medium, meaning it can be applied to nearly any type of sample. To begin, prepare one milligram of cellular protein and 500 microliters by using sonication or a French press to ly the cells to precipitate the proteins. Add one volume of tri chlorotic acid to four volumes of protein and chill on ice for 10 minutes.
Centrifuge at 12, 000 Gs for five minutes at four degrees Celsius and remove the supernatant. Then use one milliliter of ice cold acetone to Resus. Suspend the pellet before spinning a second time.
After aspirating the snat, dry the pellet in a 56 degrees Celsius heat block to denature the proteins and reduce the dye sulfide bonds. Use 500 microliters of denaturing and reduction buffer to reus. Resuspend the proteins to approximately two milligrams per milliliter.
Incubate the proteins for 30 minutes at 56 degrees Celsius, followed by 10 minutes at room temperature. Next to alkylate free sulfide groups, add 25 microliters of freshly prepared 0.3 molar IDO acetamide in water to 500 microliters of protein and incubate in the dark for 20 minutes at room temperature. After the incubation, quench the reaction by adding 10 microliters of three millimolar DTT.
Store the alkylated proteins at negative 80 degrees Celsius to digest the alkylated proteins following TCA precipitation use one molar urea in 50 millimolar heis pH 8.2 to resuspend the protein. Prepare a stock solution of Lysol, endo proteinase, or ly C in water at a concentration of two micrograms per microliter and add the enzyme to the digested protein at a final concentration of 10 nanograms per microliter samples are left at room temperature for six hours or overnight. Suspend 20 micrograms of sequencing grade trypsin in 40 microliters of 50 millimolar acetic acid and add five microliters to the lyce digestion reaction.
Incubate for six hours at 37 degrees Celsius to the alkylated proteins. Add tri Fluor acetic acid or TFA to a final concentration of 0.5%Attach a C 18 column to an extraction manifold. Then use six milliliters of aceto nitrile or a CN to wet the column.
Next, with the highest possible flow rate, use six milliliters of 80%a CN 0.1%TFA to wash the column before using six milliliters of 0.1%TFA to equilibrate the column, stop the vacuum pressure and load 500 micrograms of peptides onto the column at a flow rate of approximately one milliliter per minute. Once the peptides are bound to the column, restart the vacuum and with the highest possible flow rate, use 0.1%TFA followed by three milliliters of citric acid buffer to wash the column to carry out onco, reductive, dim methylation or ready labeling. Under a chemical hood, prepare 12 milliliters each of light and heavy ready buffers Tom Methylate peptide free.
A means add 10 milliliters of either light or heavy ready buffer to the column at a flow rate of one milliliter per minute. After applying a second 10 milliliter aliquot. To ensure labeling use six milliliters of 0.1%TFA, followed by one milliliter of 0.5%acetic acid.
To wash the column to elute the proteins, stop the vacuum and apply one milliliter of 40%to a CN 0.5%acetic acid at a flow rate of 0.5 milliliters per minute. Then add one milliliter of 80%a CN 0.5%acetic acid elute with a five milliliter syringe if desired. Mix a one-to-one ratio of heavy and light labeled peptide samples and quantify by mass spectrometry to ensure that both the and light labeling was effective fractionate the peptide mixture by first applying it to a C 18 HPLC column.
Then after using 5%a CN to wash the column for five minutes, use a gradient of five to 35%a CN over 60 minutes to elute the peptides in equal fractions. In a 96 well plate, followed by 90%A CN for one minute. After an additional four minutes of 90%A CN, use 5%a CN to re equilibrate the column.
Then combine fractions in the following way with the vacuum centrifuge. Remove the solvent from the combined fractions. Then use 130 microliters of one molar urea 0.5%TFA to Resus.
Suspend the peptides from the following fractions, and store the set of fractions at negative 20 degrees Celsius to carry out, stop and go extraction on the resuspended fractions. Prepare C 18 stage tip micro columns from 200 microliter pipette tips by using two C 18 discs with an internal diameter of 1.07 millimeter to pack them. Place the stage tips into micro centrifuge tubes and add 130 microliters of methanol and spin.
Then add 130 microliters of 80%a CN 0.5%acetic acid before spinning again after using 130 microliters of 0.1%TFA to equilibrate the tips, transfer the peptide mixture to the tips and wash. First with 130 microliters of 0.1%TFA followed by 40 microliters of 0.1%TFA. Then 40 microliters of 0.5%acetic acid to elute the peptides.
First, add 20 microliters of 40%to a CN 0.5%acetic acid. Then 20 microliters of 80%a CN 0.5%acetic acid. Combine the IITs and vacuum filtrate to dry to perform micro capillary liquid chromatography.
Tandem mass spectrometry or L-C-M-S-M-S begin by using 5%formic acid, 5%A CN to resuspend the proteins to a concentration of approximately one microgram per microliter. Apply approximately one microgram of peptides onto a 100 micrometer by 20 centimeter C 18. Reverse phase HPLC column and elute using a six to 22%gradient of a CN in 0.1 25%formic acid at a flow rate of approximately 300 nanoliters per minute, applied over 75 to 100 minutes.
Use a mass spectrometer with high resolution and high mass accuracy and identify peptides in the sample by comparing Ms.Ms Spectra raw files to a theoretical database with an algorithm such as SE quest, using the parameters shown here with the database of open reading frames in the actual and reverse orientations filter peptides to a 1%false discovery rate with a method such as the target decoy. To quantify the identified peptides, calculate the areas of heavy and light pairs of MS one extracted ion chromatograms and peptide signal to noise ratios include peptide pairs only when their average signal to noise ratio is above five. Quantify the relative abundance of a peptide in the two samples as the ratio of MS one peak areas of heavy and light versions of the same peptide calculate relative protein abundances as the median MS one peak area ratio for all peptides in the protein when filtered to a 1%peptide false discovery rate.
The ready labeling efficiency of a mix of c phyto ferment from heavy and light labeled cultures that contain 11, 194 unique peptide sequences was 98%Unfractionated visier protein lysate was similarly analyzed. Protein expression differences reproducibly reflect the ratios at which the heavy and light samples were mixed across a wide range of mixing ratios. Specifically, the fo change of 99%of the proteins was smaller than 1.6 for the one-to-one mixed samples in the one to 10 and 10 to one samples.
99%of proteins were within 3.8 fold of the expected ratio showing an increase in the standard deviation at greater distance from a one-to-one mixture. When the ready labeling was applied to the Clostridium phyto ferment proteome, more than 2000 proteins were quantified with 94%proteins measured within twofold levels for replicate cultures growing on glucose protein fold. Changes for duplicate pairs of cultures were also highly correlated.
Together these experiments support that Ready proteomics is an accurate and reproducible method to quantify protein expression differences between complex samples. Because this technique can be applied to virtually any type of sample, it paved a way for researchers in the field of proteomics to explore prote expression changes in all sorts of samples, including novel microbes, fish and mammalian cells.
