Standard Flow Multiplexed Proteomics (SFloMPro)—An Accessible Alternative to NanoFlow Based Shotgun Proteomics

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 h. 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.