This automated workflow provides a consistent protein enzymatic digestion with excellent reproducibility and throughput. This improves the accuracy and reliability of biomarker discovery, validation, and clinical application by mass spectrometry. The advantage of this technology is that the entire workflow can be completed for 96 samples in approximately five hours with intra-assay and inter-assay CV of less than 20%for the majority of proteins.
This accurate high throughput sample preparation enable us to investigate disease proteomes of tissue or biofluids on a larger scale. In addition, the automated proteomic sample preparation workflow provides a solid foundation for high content and a quantitative proteomic analysis. Demonstrating the procedure will be Dr.Qin Fu, Director of the High Throughput Center at Cedar Sinai and Mr.Casey Johnson, a research associate from my laboratory.
Before beginning the analysis, add five microliters of pooled healthy human plasma into a 96 round deep well propylene plate. When all of the samples have been loaded, open the liquid handler device software and under the method tab, select home all axes to orient the automated liquid handler. Confirm that all of the workstation syringes contain no visible air bubbles and open a new method and click Run to initiate the method.
Enter true into the enter a value to use for autosampler prompt to have an autosampler plate prepared at the end of the method and click OK.Enter one in the enter a value to use for first column prompt and click OK.Enter 12 in the enter a value to use for last column prompt and click OK.If a sample plate is being used with sample volumes of at least 20 microliters, enter true in the enter a value to use for sample plate prompt and click OK.Follow the directions in the Guided Labware Setup window and click Continue. Load the appropriate reagents into the appropriate wells and click Next. Click Next again to lay out the automated liquid handler deck as illustrated including the reagent, reaction, sample, and autosampler plates, six-well 90 microliter tip boxes, an empty 90 microliter tip box, and a full 230 microliter tip box.
Then click Finish to begin the method. At the continue after centrifugation prompt, retrieve the reaction plate and place the plate into the centrifuge. At the end of the centrifugation, return the reaction plate to its position within the automated liquid handler and click Continue.
For liquid chromatography and tandem mass spectrometry analysis, resolve the peptides in a C18 2.1 by 100 millimeter 3.5 micrometer column by high flow high pressure liquid chromatography with a flow rate of 250 microliters per minute and analyzed in line on a triple quadrupole mass spectrometer. Five minutes after loading, equilibrate the column with 5%buffer B solution and elute the peptides with a linear 5-35%gradient of buffer B over 30 minutes. At the end of the elution, wash the column with 98%buffer B for 10 minutes followed by a five-minute wash with 5%buffer B before loading the next sample.
For the online diversion, use a two-phase switching valve to divert the post-column eluent to waste before it enters the ion source. When all of the samples have been eluted, multiple reaction monitoring data can be processed. In this representative analysis, three beta-gal peptides and two albumin peptides were monitored from spiked beta-gal and processed plasma albumin proteins.
The precision of the automated sample preparation workflow was calculated as the percent of the coefficient of variance of the total proteomic selected reaction monitoring workflow minus the percent coefficient of variance of the liquid chromatography tandem mass spectrometry. As expected, good signal intensities were observed for both the human serum albumin and beta-gal proteins. To monitor the precision of the liquid transferring steps, stable isotope-labeled peptide standards can be spiked for the endogenous human serum albumin and exogenous beta-gal protein in independent reagent transferring steps.
To validate the automated proteomic sample preparation workflow, intraday coefficient of variance values were calculated from 21 wells prepared on the same day. The mean intraday percent coefficient of variance for 40 proteins ranged from 4-20%To evaluate the edge effect of the plate-based automated workflow, the percent coefficient of variance can be calculated from specific wells within designated columns and rows. In this representative experiment, the multiple reaction monitoring signal intensities were similar in all of the column and row configurations with the percent coefficient of variance ranging from 3-22%The most important steps are adding a sample correctly according to the desired 96-well plate map and correctly setting up the reagent plate according to the instruction by the software.
This automated proteomic sample preparation method allows researchers to collect reliable and quantitative proteomics data on a large scale.
