Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

Abstract

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of multiplexing up to 22 or 24 samples with tandem mass tags or isobaric N,N-dimethyl leucine isobaric tags, respectively, in a single experiment. This enhanced sample multiplexing considerably reduces mass spectrometry acquisition times and increases the utility of the expensive commercial isobaric reagents. However, the manual process of sample handling and pipetting steps in the strategy can be labor intensive, time consuming, and introduce sample loss and quantitative error. These limitations can be overcome through the incorporation of automation. Here we transferred the manual cPILOT protocol to an automated liquid handling device that can prepare large sample numbers (i.e., 96 samples) in parallel. Overall, automation increases feasibility and reproducibility of cPILOT and allows for broad usage by other researchers with comparable automation devices. as blood serum/plasma, proximal fluids, and tissues 1 , 2 .Proteomics biomarker discovery and validation have recently gained significant consideration due to the power of sample multiplexing strategies 3 , 4 . Sample multiplexing is a technique that enables simultaneous comparison and quantification of two or more sample conditions within a single MS injection 5 , 6 . Sample multiplexing is achieved by barcoding peptides or proteins from multiple samples with chemical, enzymatic, or metabolic tags and obtaining MS information from all samples in a single MS or MS/MS experiment. Among the available isobaric tags are isobaric tagging reagents (iTRAQ), commercial tandem mass tags (TMT), and in house

Evaluating cPILOT Data toward Quality Control Implementation

“…Enhanced multiplexing strategies, such as cPILOT, require robust sample preparation 27,46 and efficient data acquisition strategies 21 to ensure all reporter ions are present in each m/z scan and within and across light and heavy labeled peptides.…”

“…Samples were organized into five batches (one 20-plex and four 36-plex batches) such that each batch contained each tissue type, genotype, and age. Samples (100 μg aliquot) were dispensed into one of two 96-deep well plates according to their batch assignment, and volumes were normalized to 100 μL by the addition of 8 M urea buffer as previously described . Dithiothreitol (25 mM) was added, and both plates were placed on a mechanical rocker at room temperature for 30 min.…”

“…The S pool sample (50 μg) was dispensed across sample plates to ensure that each light and heavy group within a sample batch had two S pool samples present (hereafter referred to as QC batch ). Automated cPILOT labeling was then performed as previously described. ,, Briefly, 60 mM formaldehyde or deuterated formaldehyde and 24 mM sodium cyanoborohydride or sodium cyanoborodeuteride were added to each sample (50 μg, pH 2.5) and placed on a mechanical rocker for 15 min at room temperature to isotopically label N-termini with light [-(CH 3 ) 2 ] or heavy [-( 13 C 2 H 3 ) 2 ] dimethyl groups. Excess label was quenched by adding a 1% ammonia solution, and light and heavy groups were pooled together in a 1:1 mol ratio.…”

“…Mouse tissues were prepared according to an automated bottom-up proteomics sample preparation workflow as previously described. 27 A bicinchoninic acid protein assay was utilized to measure the protein concentration of each sample homogenate. Samples were organized into five batches (one 20-plex and four 36-plex batches) such that each batch contained each tissue type, genotype, and age.…”

“…21 Additionally, automation of sample preparation steps for cPILOT reduced analytical variability. 27 However, strategies to monitor the data quality, including maximizing the number of proteins observed across all sample channels during data acquisition, are not established.…”