A microfluidic glass chip for performing sensitive mass spectrometry (MS) is developed by integrating nanochannel-based electropreconcentration with electrospray ionization (ESI). Cation analytes in acidic buffer conditions (∼pH 3) were preconcentrated at a micro-nano-microchannel junction where a positively charged polymer, polyE-323, is coated to reverse the electroosmotic flows (anodic EOF) and prevent sample adsorption. The preconcentrated cation analytes were delivered to a monolithic spray tip using an integrated field-free electrokinetic pump, enabling a stable positive ESI with negligible dead volume. The sample preconcentration-ESI-MS achieved one order of magnitude signal enhancement in MS signal with a peptide mixture.