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Novel twist on the biotin switch cracks endothelial cell S-nitrosoproteome
However, Junmin Peng from the Emory University School of Medicine applauds the method’s quantitative nature. “[The researchers] compare two As a class of posttranslational modificatreatments, SNAP-treated versus NAPtions, few are more difficult to capture treated, and they are looking for the difthan S-nitrosylation. Created by the nonferences between the two conditions. enzymatic coupling of nitric oxide (NO) They can use a quantitative approach to to cysteine residues, S-nitrosylated proremove false positives. I think that’s the teins are notoriously finicky. In part, major advance they introduce,” he says. that’s because there are so few of them, On the other hand, Peng but S-nitrosocysteine also is says, a more sensitive mass highly labile. spectrometer (the authors Since 2001, researchers used a Q-TOF) coupled to have had the biotin switch an LC system instead of at their disposal. Whole cell 2DE might be able to protein lysates are subhandle the original biotinjected to a three-step switch protocol’s relatively chemical process that conlow protein yield. Multiplexverts S-nitrosocysteine resiing the original approach dues to biotinylated cyswith stable isotope labeling teine residues. The with amino acids in cell culresulting biotinylated proture (known as SILAC), he teins can be isolated by an adds, would reduce analysis avidin affinity step and to a single LC/MS/MS run. analyzed, revealing the “If you get 0.3 µg per petri identitysand relative dish, five dishes is 1.5 µg,” quantitationsof any S-niS-nitrosoproteomics. A modified biotin-switch protocol for detecting he says. “From that you can trosylated proteins present S-nitrosylated proteins. easily identify several-hunin the original sample. Undred proteins,” Peng says. fortunately, the method is the modified protocol, they detected Bin Huang, lead author and a postdoc not efficient enough for large-scale 15(8 S-nitrosylated spots in NAP-treated in Wang’s lab, says, “The major contrianalysis of the resulting proteins. In JPR endothelial cells and 89(17 in SNAPbution of this study is that it establishes a (DOI 10.1021/pr9005662), Danny Ling treated cells; 28 SNAP-increased S-nitrostandard operation procedure for detectWang and colleagues from Academia proteins were successfully isolated and ing trace S-nitrosoproteins with minimal Sinica (Taiwan) report a modified biotin identified with LC/MS/MS. sample and minimal technique required.” switch that circumvents these problems. “We can monitor proteins’ translaBut his method isn’t the last word on The new procedure eliminates the fition and posttranslational modification that front. A recent report in Nature Bional avidin affinity step, analyzing the simultaneously,” Wang says. “Thus, the technology describes another modificaproteins directly on five 2D gels. Total enhanced western signal of S-nitrosation that replaces the biotin with resin endothelial-cell protein extracts are retion is indeed [coming] from the post(DOI 10.1038/nbt.1545). Martı´nez-Ruiz solved on two pairs of gels: one pair reptranslational modification, not due to has been developing a modification that resenting cells incubated with the nithe difference of proteins’ translational incorporates fluorescence-based 2DE trosylating agent S-nitroso-Nlevel. It is also possible to estimate the and is described in a paper accepted for acetylpenicillamine (SNAP) and one level of S-nitrosoproteins within whole publication in Proteomics. representing cells treated with inert Ncell lysate and also the ratio of nitroso/ Besides, Martı´nez-Ruiz cautions that, acetylpenicillamine (NAP). One NAP and non-nitroso in a single protein spot.” because biotin-switch methods are one SNAP gel are then stained for total Antonio Martı´nez-Ruiz from the driven by exogenous nitrosylating agents protein content using SYPRO Ruby; the Hospital Universitario de la Princesa (such as SNAP), what emerges are only other pair is western blotted and stained (Spain), who also studies nitrosylated candidates for further study. “A preto detect biotinylated proteins. Specifiproteins, notes that for the modified sumption is that the proteins you are cally biotinylated spots are then colbiotin-switch method, “there can be nitrosylating are normally nitrosylated or lected from the fifth, preparative 2D gel, false-positive matching, because you can be nitrosylated by stimuli,” he says. digested with trypsin, and subjected to have to match the 2D signals from two “But you are forcing the reaction; the LC/MS/MS. gels, and if there are two proteins very point is that you are getting candidates The method runs the gels under nonclose [together], that can confound the for studying in more detail.” reducing conditions, preserving the analysis.” —Jeffrey M. Perkel biotin-cysteine linkage. And it uses fast-
10.1021/pr900772j
staining VisPRO dye on the preparative gel, eliminating gel fixation and improving peptide recovery and MS performance. Using the standard biotin-switch protocol on ∼1 mg of whole lysate, the authors obtained only 140 ng of S-nitrosoprotein from NAP-treated cells and 340 ng from SNAP-treated cellsstoo little for subsequent 2DE/MS analysis. But with
© 2009 American Chemical Society
Journal of Proteome Research • Vol. 8, No. 10, 2009 4393
