This study expands the capabilities for ultratrace proteomic analysis of our previous work by incorporating on-line sample desalting using a triphasic (RP/strong cation exchange (SCX)/ micro-SPE) trapping column connected to a 3.2 m x 10 pm id poly(styrene-divinylbenzene) (PS-DVB) porous layer open tubular (PLOT) column. To minimize extra sample handling steps, C18 RP packing was incorporated in the capillary tubing upstream of the SCX column for the on-line desalting. For the micro-SPE column, a 50 mu m id PS-DVB monolithic column was positioned downstream of the SCX column. High-performance separation was achieved on the PLOTcolumn at a mobile phase flow rate of 20 nL/min. The sensitivity and high resolution capability of the new multidimensional platform was evaluated using an in-gel tryptic digested sample of a cervical cancer (SiHa) cell line. For the injected amount of 1200 cells (similar to 500 ng), over 2700 peptides covering greater than 850 unique proteins were identified from the triphasic SCX/PLOT/MS analysis of a single SDS gel section (> 40 kDa). The 2-D LC/MS platform demonstrated good separation performance, such that more than 85% of the identified peptides were detected from only one salt fraction. In a triplicate analysis of the above > 40 kDa gel section, 4497 peptides and 1209 unique proteins were identified when applying stringent filtering criteria, with a false-positive rate of 2.4%. When all three SDS-PAGE gel sections of the lysed SiHa cells were analyzed, 5047 peptides and 1857 unique proteins (false-positive rate 1.8%), including cancer-related proteins such as MAP kinases, were identified.