The development of metastasis is a complex, multistep process that remains 
poorly defined. To identify proteins involved in the colonization phase of the 
metastatic process, we compared the proteome of tumors derived from inoculation 
of a panel of isogenic human cancer cell lines with different metastatic 
capabilities into the mammary fat pad of immunodeficient mice. Using a protein 
standard generated by SILAC-labeling, a total of 675 proteins were identified 
and 30 were differentially expressed between at least two of the tumors. The 
protein standard contained the proteomes of seven cell lines from multiple 
histogenic origins and displayed superior features compared to standard 
super-SILAC. The expression of some proteins correlated with metastatic 
capabilities, such as myosin-9 (nonmuscle myosin II A) and L-lactate 
dehydrogenase A, while the expression of elongation factor tu correlated 
inversely to metastatic capabilities. The expression of these proteins was 
biochemically validated, and expression of myosin-9 in clinical breast cancer 
samples was further shown to be altered in primary tumors versus corresponding 
lymph node metastasis. Our study demonstrates an improved strategy for 
quantitative comparison of an unlimited number of tumor tissues, and provides 
novel insights into key proteins associated with the colonization phase of 
metastasis formation.