We have previously reported a neutral-pH gel system buffered with Bis-Tris hydrochloride (Bis-Tris–HCl) in Zn2+–Phos-tag SDS-PAGE for advanced profiling of phosphoproteins with molecular masses of 10–200 kDa. In the current work, we describe characteristics of two neutral-pH gel systems, Bis-Tris–HCl and Tris–acetic acid (Tris–AcOH), based on comparative studies of the separation of a wide range of proteins with molecular masses from 10 to 350 kDa. For 10–200 kDa cellular proteins, the Bis-Tris–HCl system showed a higher resolving power in a 2-D fluorescence DIGE analysis of certain phosphoproteins, e.g. histone H3 (15 kDa) and elongation factor 2 (95 kDa). Furthermore, there was a large difference in the 1-D migration patterns of phosphorylated species of extracellular signal-regulated kinases 1 and 2 (ERK1/2, 44/42 kDa), which arise from changes in the phosphorylation status of the Thr-202 and Tyr-204, in the two buffer systems at the same concentration of Zn2+–Phos-tag. In contrast, shifts in the mobility of various phosphorylated species of a high-molecular-mass protein, ataxia telangiectasia-mutated kinase (ATM, 350 kDa), could only be detected in the Tris–AcOH system with a 3% w/v polyacrylamide gel strengthened with 0.5% w/v agarose.