The design of proteome projects is often restricted by the available amounts of sample, and thus there has been much effort to date to improve the sensitivity of proteome analyses. We have developed a new ultrasensitive nanoLC-MS system for the proteome analysis of microscale samples using a nonporous reverse phase column. Although nonporous particles have low binding capacity due to low functional group density, we found that the use of C30 particles compensated for this disadvantage and nanoLC-MS proteome analyses of microscale samples using a C30-packed column exhibited superior performance compared to the use of porous C18 columns. This system enabled us to identify 3278 and 1407 proteins from 1000 and 100 HEK293F cells, respectively. The results showed that the use of nonporous columns solved problems intrinsic to the analysis of microscale protein samples.