We demonstrate matrix-free pH gradient electrofocusing of proteins within an 85 nm deep nanochannel. In contrast to conventional isoelectric focusing where the fluid does not move, this pH gradient method traps protein molecules flowing through a channel by balancing electric forces due to pH-dependent protein charge and viscous drag forces caused by electro-osmosis. The nanoscale depth of the device and the low voltage used limit convection relative to diffusion, thus producing a stable focused band of protein. R-Phycoerythrin (RPE) and Dylight labeled streptavidin (Dyl-Strep) were focused within a nanochannel using applied voltages between 0.4 and 1.6 V. Concentration enhancement factors of over 380 have been achieved within 5 min. Varying the buffer pH (between 2.7 and 7.2) at the boundaries of the nanochannel affected the shape of the focused bands. For RPE, a pH span of 4.5 (pH 2.7 to 7.2) yielded the narrowest peak while a span of 2.4 (pH 2.7 to 5.1) produced a significantly wider peak. Such matrix-free nanofluidic devices with pH gradient electrofocusing may enable on-chip integration of orthogonal separation techniques with mass spectrometry offering labor savings and enhanced performance.