PT - JOURNAL ARTICLE AU - Conor Jenkins AU - Ben Orsburn TI - Standard Flow Multiplexed Proteomics (SFloMPro) – An Accessible and Cost-Effective Alternative to NanoLC Workflows AID - 10.1101/2020.02.25.964379 DP - 2020 Jan 01 TA - bioRxiv PG - 2020.02.25.964379 4099 - http://biorxiv.org/content/early/2020/02/25/2020.02.25.964379.short 4100 - http://biorxiv.org/content/early/2020/02/25/2020.02.25.964379.full AB - Multiplexed proteomics using isobaric tagging allows for simultaneously comparing the proteomes of multiple samples. In this technique, digested peptides from each sample are labeled with a chemical tag prior to pooling sample for LC-MS/MS with nanoflow chromatography (NanoLC). The isobaric nature of the tag prevents deconvolution of samples until fragmentation liberates the isotopically labeled reporter ions. To ensure efficient peptide labeling, large concentrations of labeling reagents are included in the reagent kits to allow scientists to use high ratios of chemical label per peptide. The increasing speed and sensitivity of mass spectrometers has reduced the peptide concentration required for analysis, leading to most of the label or labeled sample to be discarded. In conjunction, improvements in the speed of sample loading, reliable pump pressure, and stable gradient construction of analytical flow HPLCs has continued to improve the sample delivery process to the mass spectrometer. In this study we describe a method for performing multiplexed proteomics without the use of NanoLC by using offline fractionation of labeled peptides followed by rapid “standard flow” HPLC gradient LC-MS/MS. Standard Flow Multiplexed Proteomics (SFloMPro) enables high coverage quantitative proteomics of up to 16 mammalian samples in about 24 hours. In this study, we compare NanoLC and SFloMPro analysis of fractionated samples. Our results demonstrate that comparable data is obtained by injecting 20 μg of labeled peptides per fraction with SFloMPro, compared to 1 μg per fraction with NanoLC. We conclude that, for experiments where protein concentration is not strictly limited, SFloMPro is a competitive approach to traditional NanoLC workflows with improved up-time, reliability and at a lower relative cost per sample.Data are available via ProteomeXchange with identifier PXD016704.Abstract Graphic