We developed a novel application to conduct pseudopodia proteomics. Pseudopodia 
are ventral actin-rich protrusions and play functional roles in cell migrations. 
Identification of pseudopodia proteins leads to a further understanding of 
malignant phenotypes of tumor cells and novel therapeutic strategies. In our 
application, tumor cells were placed on a fibronectin-coated porous membrane to 
form pseudopodia. According to the motile potentials of the cells, the cells 
formed pseudopodial microprocesses in the pores. An excimer laser, which was 
used for ophthalmic refractive surgeries, horizontally ablated cells at the 
membrane surface to remove the cell body. The microscopic observations and the 
protein expression studies suggested that the laser treatment caused no apparent 
damages to pseudopodia. Proteins in whole cells and pseudopodia fractions were 
individually solubilized, labeled with a highly sensitive fluorescent dye, and 
separated using two-dimensional difference gel electrophoresis. Among 2508 
protein spots observed, 211 had different intensity between whole cells and 
pseudopodia fractions (more than fourfold differences and P-value of <0.05). The 
protein enrichment depended on the pore size. Mass spectrometric protein 
identification revealed 46 pseudopodia-localizing proteins. The localization of 
novel pseudopodia-localizing proteins such as RAB1A, HSP90B, TDRD7, and vimentin 
was confirmed using immunohistochemical examinations. The previous studies 
demonstrated that these four proteins may function in the cell migration 
process. This method will provide insights into the molecular details of 
pseudopodia and a further understanding of malignant phenotypes of tumor cells 
and novel therapeutic strategies.