progress has been made in defining growth factor conditions that support self - renewal [ 44 - 46 ], and hesc lines have been isolated in the absence of mouse embryonic fibroblasts and in animal protein free culture conditions [ 47, 48 ]. a more refined understanding of the biology of hescs has contributed the development of a defined medium utilizing ligands for igf1r and erbb2 / 3 receptors to promote in self - renewal ( tcs and ajr, submitted ). we and others have performed transcriptional analyses of hescs, using cdna and oligo nucleot ide microarrays, sage, mpss and est enumeration. these techniques have enabled the collation and comparison of transcriptional profiles from multiple hesc lines and their differentiated derivatives and have highlighted an expanded set of hesc specific markers and signaling pathways that may regulate self - renewal or differentiation. using pathway analysis we were also able to identify key pathways that are active in escs ( reviewed in [ 16 ] ). while these efforts have been highly valuable in defining the transcriptional profile of undifferentiated hescs, they are only predictive of translation and do not shed light on post - translational events in this unique cell type. these processes may also be highly regulated, which could contribute significantly to the overall conversion of genetic information to actual protein function. we report here a proteomic analysis of pluripotent hescs by using two large - scale western blotting systems and highlight post - translational events in undifferentiated hescs. the expression of 545 bands was detected, potentially representing 529 proteins, or their migratory isoforms. in addition, one hundred and forty phospho - specific antibod ies were used to identify 85 different phosphorylated sites, on 76 proteins in these cells. the detected proteins were annotate d into functional classes representing diverse cellular processes. for example, multiple proteins were detected that have been suggested to regulate the pluirpotent state in mouse escs or hescs. defining the interplay of these multiple signaling pathways will be critical in understanding the self - renewal versus differentiation decisions of hescs. therefore, our data provide a powerful framework for the functional analysis of specific proteins, protein classes, or molecular pathways. in particular, the availability of antibod ies for candidate proteins is a major benefit of this approach compared to 2d - gel or hplc - ms / ms based proteomics. although these western blotting approaches are currently more limited in scope than most large - scale cdna based assays, detecting up to 1000 proteins compared to tens of thousands of transcripts, they have the potential to highlight translational events and post - translational modifications. by comparison, sage and mpss are limited to detecting short sequence " tags " adjacent to the poly - a tail of transcripts, and may not disting uish splice variants with the same 3 ' exon. we detected 42 proteins with multiple closely migrating bands ( fig. 3c ), suggestive of closely related isoforms or post - translational modifications such as phosphorylation. these focused proteomic approaches are therefore likely to be highly complimentary to transcriptional analyses in investigating the functional expression of the genome in hescs and during cellular differentiation. one potential issue with this approach is that multiple antibod ies are included in each lane, which could possibly lead to misidentification of bands. to demonstrate that identified proteins were expressed in hescs, the same antibod ies used in the powerblot assay were used to confirm expression of 10 representative proteins by immunoflu orescence ( fig. 4 ). furthermore, 13 proteins were detected with multiple different antibod ies, and 35 proteins ( table 1 ) were detected in both the powerblot and kinexus assays. this provided internal, or independent, confirmation of expression of these proteins. other studies have also demonstrated the expression of several of the proteins we detected in hescs. these include oct4, a key marker of the pluripotent state, connexin 43 and gsk3β, confirming the reliability of large - scale western blotting. finally, several proteins detected by our assays were also detected in hescs by ms approaches including karyopherin α [ 19 ]. additionally, the powerblot assay was performed in duplicate, and was shown to be highly reproducible. this suggested that this approach should be informative when comparing hescs to their differentiated derivatives. two candidate proteins, tnik and p130 cas, were downregulated, or exhibited altered localization upon spontaneous differentiation of hescs, respectively. this indicated that they were novel markers of undifferentiated cells and molecules that could be functionally involved with self - renewal. it is impossible in an initial manuscript to analyze and rigorously test all the predictions that could be made from comparing transcriptional and proteomic data sets. however, we did examine key features to illustrate the power of this methodology. potential new markers for hescs were identified, the expression and activation of proteins in key self - renewal pathways were confirmed, and a diverse range of proteins were detected and expression correlated with transcriptional analyses. in addition, we highlighted several candidate signaling pathways that may be relevant to self - renewal. examination of tight junction protein expression indicated that undifferentiated hescs could form polarized epithelia, which has also been recently suggested by ultrastructural analyses [ 49 ]. discrete localization of erbb3 may also suggest basolateral separation of this receptor from soluble ligand. these analyses highlight that predictions from a combination of transcriptional and proteomic approaches will serve to focus the investigation of hescs in the future. in summary, we generated a focused proteome of hescs using large - scale western blotting and sorted the detected proteins according to function and signaling pathways. this characterization provides important basic information on expressed proteins, their isoforms and post - translational modifications, and tools for the continued investigation of the underlying molecular characteristics of hescs. importantly, we provide a list of tools, in the form of commercially available antibod ies, which can be used to interrogate the function of these molecules in self - renewal or differentiation. culture of human embryonic stem cellsfor the powerblot analysis, enzymatically passaged bg01 hescs were grown as described previously [ 23 ]. these conditions were necessary to scale up the culture to generate the milligram amounts of protein lysate required for this analysis. these conditions maintain cell populations that express the appropriate markers of pluripotency and can differentiate to representatives of all three germ layers, but may lead to eventual accumulation of trisomies for chromosomes 12, 17 or x [ 26 ]. for the kinexus assays, bg03 hescs were maintained in mef - conditioned medium ( mef - cm ) without the accumulation of karyotypic abnormalities as described previously [ 14, 26 ]. hescs were also maintained in a defined medium as indicated. these conditions are described in detail elsewhere ( tcs, ajr, submitted ). briefly, the media consisted of dmem / f12 ( invitrogen ), 2 % fatty acid - free cohn ' s fraction v bsa ( serologicals ), 1× nonessential amino acids, 50 u / ml penicillin / streptomycin, 50 μg / ml ascorbic acid, 10 μg / ml bovine transferrin, 0. 1 mm β - mecaptoethanol ( all from invitrogen ), 1× trace elements a, b & c ( mediatech ), 10 ng / ml hergulin1β ( peprotech ), 10 ng / ml activina ( r & d systems ), 200 ng / ml lr3 - igf1 ( jrh biosciences ), and 8 ng / ml fgf2 ( r & d systems ).