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RESEARCH PROFILES
Enrichment of plasma membrane proteins from pluripotent cells
further analysis by shielding proteins and preventing proteolytic digestion later on,” explains Krijgsveld. Reduction and alkylation unfolds the proteins, Recently, much media attention has which are digested in a three-step been focused on the possibility that process with two enzymes. Why three human embryonic stem cells (hESCs) digestion steps? “We started off with could be used to treat various illnesses. a simple tryptic digestion and got a However, when hESCs are cultured for number of peptides, but then we started long periods of time, they can develop analyzing the residual fraction of prochromosomal abnormalities similar to teins that were not digested in the first those observed in human embryonal step,” he says. “Then, when we applied carcinoma cells (hECCs), which are a second enzyme, for instance, Lys-C, pluripotent cells that originate from that yield went up dramatically.” germ cell cancers. Thus, these In the final version of the propotentially therapeutic cells (a) (b) tocol, proteins are digested first could pull a “Dr. Jekyll and with Lys-C, which generates large Mr. Hyde” switch and cause fragments. Then, two steps of cancer. To differentiate bona trypsin digestion are performed. fide hESCs from those cells in Finally, peptides are analyzed by the culture that have become LC/MS. malignant, Jeroen Krijgsveld and Of the >1000 proteins identified co-workers at Utrecht University in each cell line, about half were and Hubrecht Institute (both known or predicted to be memin The Netherlands) report in Pretty green coat. Connexin43 (green) is expressed on (a) brane proteins. In particular, JPR (2008, 7, 2936–2951) on the the plasma membranes of hESCs but not on (b) the surfaces 237 and 219 plasma membrane development of a new method of hECCs. Nuclei are visualized by blue staining. Scale bars = proteins were identified in hESCs to enrich the plasma membrane 20 μm. and hECCs, respectively, which proteins of both types of cells. is >10× more than were identiWith the method, they confied when the researchers performed a kept all of the cellular membranes in ducted a proof-of-principle study on traditional solubility-based technique the initial sample but optimized the small numbers of cells and discovered on these cells. Also, Krijgsveld says that digestion conditions for plasma memproteins that appeared to be unique to considerably fewer cells were required brane proteins. They also kept track of hESC or hECC surfaces. for this method than are necessary for the numbers of plasma membrane proSome researchers have compared more conventional approaches. A total teins and other membrane proteins to hESCs with hECCs to figure out the of 152 plasma membrane proteins were determine the degree of enrichment. factors that cause hESCs to become unique to either hESCs or hECCs. For In the method, cells undergo a soft malignant, but Krijgsveld says that example, different fibroblast growth mechanical lysis to break them open these initial reports lack characterizafactor receptors were expressed in the and remove nuclei. The sample is centions of the cells’ plasma membrane two cell types. Because antibodies trifuged at a low speed to get rid of large proteomes. Scientists may have shied against many proteins are difficult to cellular structures. Next, membranes away from such studies because plasma come by, only a few of the identified are spun at a high speed on a sucrose membranes are difficult to separate proteins could be validated by western cushion to prevent them from pelleting from other membranous intracellular blotting and immunofluorescence. and to make resuspension easier. “So, structures. In addition, pluripotent cells The current study was designed basically, proteins stay in solution, but present unique challenges. These cell solely to develop and optimize the enare still in a kind of pellet in a 60% sulines are so difficult to come by that richment procedure, so Krijgsveld and crose cushion,” explains Krijgsveld. The sample volumes are limited. Also, cellco-workers are gearing up to use the membranes are washed with sodium surface-labeling techniques for plasma method for a quantitative comparison carbonate to remove proteins that are membrane isolation don’t work well of plasma membrane proteins from loosely associated. with hESCs and hECCs; that’s because hESCs and hECCs. In addition, they The remaining integral membrane these cells form multiple layers in a plan to examine proteins from cells that proteins are deglycosylated with petri dish, and only the top layer of cells are at various stages of differentiation. peptide-N-glycosidase F (known as would be labeled. —Katie Cottingham PNGaseF). “Glycosylation could prevent By performing conventional plasma membrane enrichment techniques on hESCs and hECCs, Krijgsveld and co-workers identified only a handful of proteins. After that, they knew they had to develop their own strategy. “We combined many individual steps that have been described previously in other studies, but we tried to combine the components in the eventual protocol [so that we would obtain] the highest number of proteins,” says Krijgsveld. The team took a less restrictive approach than other researchers have taken: they
2590 Journal of Proteome Research • Vol. 7, No. 7, 2008
© 2008 American Chemical Society
