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Mann, Colleagues Use SILAC to ID 5,111 Proteins in Mouse Embryonic Stem Cells

A team of researchers in Germany has identified more than 5,000 proteins in mouse embryonic stem cells, the most comprehensive protein map of a stem cell to date and nearly three times what had been reported in previous studies.
In an article published in the April edition of the journal Molecular & Cellular Proteomics, the authors report using stable isotope labeling by amino acids in cell culture, or SILAC, to achieve their results. Originally published in November, the journal reran the article as part of this month’s issue highlighting posters that were included and work that was discussed during the 8th International Symposium on Mass Spectrometry in the Health and Life Sciences held last summer [See PM 09/06/07].
The study is notable for two reasons: the 5,111 distinct proteins identified make up the most comprehensive protein map of a stem cell to date, and the researchers’ success in using SILAC to fully label murine embryonic stem cells when they are grown feeder-free during the last phase of cell culture.
Prior to the work described in MCP, the most comprehensive map of the mouse embryonic stem cell proteome contained 1,790 proteins, though that experiment was non-quantitative, therefore making differential analysis impossible, the authors of the MCP study said.
Another study, which identified 1,775 proteins, was quantitative, but used peptide counting, “a method suitable for highlighting large scale changes in protein abundance but not appropriate for determining accurate quantitative changes on a protein-by-protein basis,” the authors said. “This is especially true for low-abundance level, regulatory proteins.”
Methods using stable isotopes provide more accurate quantitation, and metabolic labeling has the added feature of being able to eliminate error-prone parallel steps in protein purification protocols. But metabolic labeling methods have been used mainly for transformed cell lines, “and labeling of ES cells, a cell type that is difficult to culture, has not yet been demonstrated,” the researchers said. 
Using SILAC, they show that complete metabolic labeling of murine embryonic stem cells is possible. Included in the proteins they identified were low-abundance protein classes such as transcription factors and kinases and “well-documented stem cell markers, which suggests that the SILAC-labeled cells retain stemness,” or the undifferentiated and pluripotent state.
While in large part a proof-of-principle paper, the MCP study “also establishes some important facts about ES cells,” Matthias Mann, a professor at the Max Planck Institute of Biochemistry and the corresponding author on the study, said in an e-mail to ProteoMonitor.
“First that they have a very complex proteome — there was a feeling with some researchers that ES cells only need a complex transcriptome, [but] not a complex proteome. Secondly, we found very good correlation with activating and repressive chromatin marks. Basically, whenever we found the protein there was an activating mark at the genome level … and we don’t find the protein if there was a repressive mark,” he said. “This may sound obvious, but is actually controversial in the field.”
He and his fellow researchers also found that an off-gel method works as well as “or better than cell fractionation combined with in-gel digestion,” Mann said.
He and his colleagues first tested whether mouse embryonic stem cells could grow in SILAC medium using feeder cells or under feeder-free culturing conditions. For this they used mouse embryonic stem-cell lines R1 and G-Olig2.

Mann and his colleagues “plan to use the SILAC ES cell system for studying differentiation of ES cells and particularly to see how close the proteomes of ES cells [are to] the inducible ES cells, [which] are capturing everybody’s imagination.”

Embryonic cells are usually cultured on mouse embryonic fibroblasts inactivated by irradiation or mitomycin C. “The feeder layer is renewed when passaging ES cells and may represent a substantial source of unlabeled amino acids,” the authors said. When they evaluated this method, they found that the stem cells could be SILAC-labeled, but the feeder cells interfered with labeling efficiency.
“The low labeling efficiency of 0.83 and the bimodal distribution of peptide ratios suggest that the sample is composed of partially labeled feeder cells and of fully labeled ES cells,” the researchers said. “Likely even low contamination with feeders has a strong contaminating effect because their diameter is approximately twice that of ES cells.”
Having failed at that try, they then grew stem cells in BMP4-supported feeder-free culture for three passages prior to harvest. This resulted in unimodal distribution of high incorporation ratios of heavy amino acids and an average labeling efficiency after five passages of 97 percent, “showing that mouse ES cells can be efficiently and completely SILAC-labeled.”
With the compatibility of embryonic stem cell culture established, the researchers then set out mapping the proteome by two methods. The first was by standard subcellular fractionation. Three fractions were achieved: cytoplasmic, nucleoplasmic, and chromatin/membrane. The fractions were separated by 1D SDS gel. The gel lanes were sliced into 15 gel blocks and in-gel digested then analyzed by LC-MS/MS.
The combined analysis of 45 gel slices resulted in 516,649 tandem mass spectra, which yielded 35,963 unique peptide identifications and 4,036 distinct proteins, mapping to 3,931 locations in the mouse genome.
The team then did an analysis by isoelectric focusing of peptides, choosing Agilent’s OFFGEL Fractionator.
In-solution digested embryonic stem cell extracts were fed into the instrument and peptides were separated for 50 kilovolt-hours, resulting in 264,372 tandem mass spectra. The authors identified 27,362 unique peptides with an average absolute mass accuracy of 559 ppb. A yield of 3,972 proteins, mapping to 3,892 different Ensembl entries, were achieved.
The data from the two experiments were then combined and analyzed “to arrive at a high confidence proteome of mouse ES cells.” The raw mass spec files were imported into the MaxQuant software and analyzed “as a whole using uniform statistical criteria, in particular the requirement of two fully tryptic peptides in the correct SILAC states with very low mass deviation and a 99 percent certainty of identification at the protein level as assessed by reverse database searching,” the authors said.
This resulted in 781,021 tandem mass spectra correlating to 49,445 unique peptide sequences. This yielded a mouse embryonic stem-cell proteome of 5,111 proteins, mapping to 4,972 distinct locations in the mouse genome.
Checking for the presence of known stem cell markers, Mann and his colleagues found OCT4 with seven peptides, SOX2 with nine peptides, and NANOG with two peptides.
“These three ‘master regulators’ are intimately involved in the maintenance of stemness, and loss of their expression is concomitant with exit from the pluripotent state,” the authors said.
They did not detect known markers SALL4, DPPA2, or DPPA4, however. The authors attribute this to their low abundance.
They also found 156 protein kinases and 131 transcription factors, 4.1 percent and 3.5 percent, respectively, of all proteins identified. “Taken together, these observations suggest that we covered the mouse ES cell proteome in considerable but not yet complete depth,” the researchers said. Using less-stringent criteria on their data than that used to identify the 5,111 proteins, they were able to detect the presence of at least 1,000 additional proteins, they report. “Thus further technology development is still needed for more comprehensive coverage of the ES cell proteome,” they said.
While the amount of proteomics-directed research into stem cells is still relatively limited in number that may be changing. Last spring, the Human Proteome Organization announced an initiative to spur greater scientific research into stem cells using proteomic technologies [See PM 06/21/07 and 10/11/07].
Building on his and his colleagues’ work, Mann said they “plan to use the SILAC ES cell system for studying differentiation of ES cells and particularly to see how close the proteomes of ES cells [are to] the inducible ES cells, [which] are capturing everybody’s imagination.”

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