PNNL

Abstract

Multidimensional separations can greatly increase the depth of coverage in proteome profiling. However, a major challenge for multidimensional separations is the requirement of large biological samples, often containing milligram amounts of protein. We have developed nanowell-mediated two-dimensional (2D) reversed-phase nanoflow liquid chromatography (LC) for in-depth proteome profiling of low-nanogram samples. Peptides are first separated using high-pH reversed-phase LC and the effluent is concatenated into 4 or 12 nanowells. The contents of each nanowell are reconstituted in LC buffer and collected for subsequent separation and analysis by low-pH nanoLC-MS/MS. The nanowell platform minimizes peptide losses to surfaces in offline 2D LC fractionation, enabling >5,800 proteins to be confidently identified from just 50 ng of HeLa digest. Furthermore, we demonstrated deep proteome profiling of small populations of cells including ~650 HeLa cells and ~10 single human pancreatic islet thin sections (~1000 cells) from a pre-symptomatic type 1 diabetic donor