Aliquoting of isobaric labeling reagents for low concentration and single cell proteomics samples

The introduction of isobaric tagging reagents enabled more accurate, high-throughput quantitative proteomics by enabling samples to be multiplexed. One drawback of these workflows is the relative expense of the proprietary chemical reagents, which is often only second to the expense of the instruments themselves. These highly reactive chemical tags are only commercially available in relatively large aliquots compared to the typical amounts of peptides analyzed in proteomic workflows today. Excess reagents are typically disposed of following labeling of small batches or within a few weeks of opening. We present a simple procedure to aliquot commercial isobaric tagging reagents and demonstrate the successful and high efficiency labeling of multiple samples over a period of six months. The samples presented herein were selected as the most diverse samples labeled by prepared aliquots from a single labeling reagent kit. We observe comparable labeling efficiency from 100 microgram to 100 picograms of peptide when labeling samples from both human digest standards, cancer cell lines prepared in-house and from cells directly obtained from organ donations, despite differences in cell type, lysis and digestion. No labeling experiment of whole human proteomics samples achieved less than 92% labeling efficiency over this period. When preparing phosphoproteomic samples from a cancer cell line digest at approximately 6 months from the date of the aliquoting procedure, we observed a decrease in labeling efficiency to approximately 86%, indicating we are approaching the end of the useful lifetime of these prepared aliquots. Over this period, we have effectively reduced the reagent costs of each labeling experiment to less than 10% of the predicted costs when following the manufacturer instructions for use and disposal. While aliquoting of reagents can be performed by hand, we provide a complete template for automatic aliquoting using an affordable liquid handling robot, including plans for 3D printing of parts we have found useful for streamlining this procedure.