Protein Phosphorylation Cytometry Via Single Cell Isoelectric Focusing

Cellular processes are profoundly affected by chemical modifications of proteins. Perhaps the most important chemical modification is protein phosphorylation, which alters the activity, function, regulation, and degradation of a protein. This broad swath of impact all derives from the simple post-translational addition (or subtraction) of a negatively-charged phosphate group. Given the importance of phosphorylation, it is striking that substantial measurement challenges stymie progress, especially with cellular resolution. Detection specificity (selectivity) is chief among the measurement challenges. When a phospho-specific immunoreagent is available as the basis of an immunoassay, cross-reactivity and off-target binding limit selectivity and multiplexing [2]. Consequently, we advance IEF [1] to measure protein phosphorylation in individual cells.

With single-cell resolution, we profile developmental variation among nominally ‘homogeneous’ stem cell populations. Through judicious sample preparation, we utilize native and denaturing single-cell IEF to profile both phosphorylated and unphosphorylated protein forms. The rapid (minutes) IEF separation resolves multiple protein species within each cell, down to single-charge unit differences, as is essential for phospho-profiling. After reporting assay and device design, as well as assay characterization, we will overview application of single-cell ‘phospho-proteomics’ to quantitatively assess and then optimize cell maturation stages within a heterogeneous, stem-cell-derived, terminally-differentiated cell population.

Taken together, this targeted ‘phospho-proteomics’ assay brings the selectivity of IEF to individual cells, as is central to developmental, disease, and life processes.

References:

1. Tentori AM, Yamauchi KA, Herr AE. submitted 2016

2. Bordeaux J, et al. Biotechniques 2010.