We present here an in-depth proteomic analysis of ovarian cancer cell lines and ovarian cancer tumor cells, including separate analyses of cell surface and secreted proteins. The study was based on intact protein fractionation followed by trypsin and LC-MS/MS 
, allowing sensitive detection of low abundance proteins. Altogether, we generated an extensive profile of proteins consisting of more than 6,500 unique identifications with an error rate less than 1%. A recent study of ascites fluid consisting of proteins derived from mixed populations of cells 
identified 2,737 proteins. 2,307 (84%) of the proteins reported in that study were encompassed among proteins identified in our study.
The use of spectral counts as a measure of abundance coupled with comparisons of proteins found in the cell surface or extracellular compartments and in whole cell lysate profiles, allowed identification of proteins enriched in particular compartments. Peptide counting provides abundance estimates that correlate reasonably well with those determined by other methods 
. In our study we also observed a good correlation between estimates of abundance of tissue inhibitor of metalloproteinase 1 (TIMP1) and epithelial cadherin (CDH1) based on spectral counting and assessment based on Western blot analysis. Isotopic labeling with SILAC 
also provided an efficient means to distinguish proteins in conditioned media that are released from cultured cells from proteins contributed by bovine serum in the culture medium.
Our analyses have provided insight into the contribution of surface protein shedding and release to the protein composition of the extra-cellular compartment. Proteins highly enriched in culture media represent a potential source of circulating markers for ovarian cancer. Transcripts corresponding to some of these proteins (e.g., KLK6, 7 and 9) are relatively abundant in ovarian tumors compared to most normal tissues 
. There is already evidence for the occurrence of most of these proteins in normal human plasma. Of the 60 proteins listed in Table S2
, 48 (80%) were present in the Human Plasma Peptide Atlas 
. However, to be effectively detected at increased levels and as expected of a biomarker candidate, a protein should have high secretion rates specifically by tumor cells, low concentration in normal plasma and also a low clearance from the circulation. Proteins that we identified in the conditioned medium (Table S2
) whose corresponding mRNAs are relatively highly expressed in ovarian cancer tissue include TIMP1, IGFBP3 (insulin-like growth factor-binding protein 3), MDK (midkine), PROS1 (vitamin k-dependent protein s) and SLPI (secretory leukoprotease inhibitor). Several other proteins that have been proposed as potential biomarkers for ovarian cancer were identified in the extracellular compartment of cells analyzed in this study, including WFDC2 (HE4) and MUC16 (mucin 16, CA125), a membrane glycoprotein that was significantly enriched in conditioned media from CaOV3, OVCAR3 and ascites cancer cells and which is used as a marker of ovarian cancer 
. These findings suggest that other proteins enriched in the culture media and/or on the cell surface may have potential as markers for ovarian cancer.
We observed a substantial enrichment of CDH1 both on the cell surface and in culture media, likely as a result of a shedding process driven by metalloproteases 
. Shed extracellular domains of proteins have potential utility as circulating biomarkers, as has been suggested for some cadherins 
. Other soluble fragments of membrane proteins, which we found in the extracellular compartment, notably MSLN (mesothelin) have also been proposed as potential biomarkers 
The process of surface protein shedding as observed in ascites derived tumor cells is relevant to tumor invasion and metastasis 
, as exemplified by the role of cadherins. Members of this family involved in adherent junctions and desmossomes include desmogleins (DSG1 and DSG2) and the recently discovered family of calystenins (CLSTN1 and CLSTN3). Interestingly, calystenins are localized in the postsynaptic membrane and proteolytically cleaved N-terminal fragments corresponding to the extracellular domain have been observed. Proteolytic cleavage of the extracellular domain of calystenins is known to impact directly Ca2+ signaling in brain 
The dynamics of shedding of cell surface proteins and release into circulation (or conditioned media as detected here) has relevance to diagnostics and imaging. However their reduced representation on the cell surface and occurrence in circulation diminishes their utility as imaging or therapeutic targets compared to stable, non-shed proteins. For example, integrin alpha 6 (ITGA6) is significantly enriched on the cell surface of all three ovarian cell lines as well as the ascites tumor cells, is relatively highly expressed in ovarian cancer and was not detected in conditioned media (see Table S4
Our findings indicate that the serous ovarian cancer derived cell lines OVCAR3 and CaOV3 are good models of serous ovarian cancer to identify potential biomarkers given the similarities we have observed with tumor cells isolated from ascites from a patient with serous ovarian cancer. Our data also indicates extensive cell surface protein shedding in tumor cells obtained from ascites. The extensive proteomic profiling of key compartments for the four ovarian cancer cell populations that we have studies provides a rich dataset for further exploration and integration with other data for identification of potential circulating markers and imaging and therapeutic targets.