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Using this approach, Valenzuela et al.report the generation of 200 targeted muta-tions, mostly with fairly large deletions of thetargeted genes. They also report interestingand specific gene-expression patterns of theinserted β-galactosidase reporter aftergermline transmission, in line with many pre-vious experiments with targeted or gene-trapinsertions with reporter constructs. Becausemany of the mutations involve large dele-tions, presumably including intron sequenceswhere regulatory elements may reside, cau-tion should be applied in assuming that thegene-expression patterns observed reflect thefull endogenous gene expression pattern.

Large deletions are not the only muta-tions useful for a community resource.Perhaps the most useful resource would be alibrary of ES cells containing a set of ‘floxed’alleles across the genome. In such muta-tions, a key exon is flanked by loxP sites,such that expression of the Cre recombinaseenzyme causes gene deletion. Crossing withappropriate Cre-expressing lines can producenull, tissue-specific and inducible loss-of-function mutations6. The rapid BAC-basedapproach espoused by Valenzuela et al. is animportant first step in high-throughput tar-geted mutagenesis and challenges others todesign strategies able to generate multiplekinds of targeted mutations in a timely andcost-effective manner.

When genome sequence centers wereestablished, cost-effectiveness was a majorcriterion for success—the cost of sequencingper base was measured and evaluated. Shouldwe not apply the same criterion to mutationgeneration? Many of the costs of mouse or ratmutagenesis come from the costs of breedinganimals, which vary widely from center tocenter. However, we can try to evaluate thecost of making and identifying different kindsof mutations and estimate the cost of saturat-ing the genome. Zan et al. estimate a cost of$18,000 per mutation for their ENU/yeasttruncation assay. Assuming 40,000 genes inthe genome, this would amount to $720 mil-lion to find all mutations. Similar costs proba-bly apply to current sequence-based screeningapproaches in mouse ES cells. By consultinggene-trap centers on their current fundingand their projected output, I have estimatedthe direct cost of generating one RACE-tagged gene-trap insertion in ES cells at about$200. Since this approach is random, genomesaturation would require a library of at least120,000 clones, giving a total cost of $24 mil-lion. It would be interesting to get an estimateof the per gene direct cost of the approachfrom Valenzuela et al. to begin a comparativecost-benefit analysis.

Once mutations in all the genes are avail-able in ES cells or sperm archives, the cost ofmaking mutant animals becomes the limit-ing feature. This problem has been alleviatedto some extent by the recent development ofrobust hybrid mouse ES cells that can bemade directly into mice by introduction intotetraploid host embryos7. This method isalso particularly useful when multiple muta-tions need to be studied in the same mouse,because these can be introduced into one EScell and made directly into mice withoutgenerations of intercrosses. We can look for-ward to a future where the generation of

NATURE BIOTECHNOLOGY VOLUME 21 NUMBER 6 JUNE 2003 627

Carbohydrate modifications can pro-foundly affect protein function. Theirimportance in disease is evident from agrowing number of embryonic lethal phe-notypes seen in knockout mice with defectsin glycoconjugate assembly or processing.In humans, the list of congenital disordersof glycosylation (CDG)1,2 is also lengthen-ing, with impaired glycosylation resultingin severe mental and physical problems for hundreds of patients worldwide.Identifying specific mis-glycosylated pro-teins that cause these devastating condi-tions is far from simple because not allglycoproteins are implicated in CDG. Inaddition, analyzing the glycosylation stateof large numbers of proteins is problematicbecause unglycosylated peptides outnum-ber glycosylated ones. In this issue, twopapers describe innovative methods toidentify proteins with carbohydrates linkedto asparagine residues (N-glycosylated pro-teins). The methods involve replacing thesugar chains on peptides with tags and thenidentifying the peptides using mass spec-trometry (MS)3,4.

Glycans on proteins can be either N-linked or O-linked (on the hydroxyl group

of serine or threonine). In humans, mostknown CDGs result from N-glycosylationdefects; only a few defects are known in O-glycosylation. N-glycosylated proteinsare found in secretions, on the cell surface,and in the endoplasmic reticulum (ER),Golgi and lysosomes. Newly synthesizedproteins entering the ER can be glycosy-lated on asparagine residues when theamino acid appears in an Asn-X-Ser/Thrconsensus sequence, where X can be any-thing except proline. Some ‘X’ residues aremore permissive than others, and site occu-pancy can be cell-type specific5. Rarely, cys-teine substitutes for serine/threonine.

Although an Asn-X-Ser/Thr might be a‘potential glycosylation site,’ this does notnecessarily mean that the site is actuallyoccupied. In fact, only about two-thirds ofpotential sites are used6. Using mass spec-trometry to identify occupied glycosylationsites is cumbersome because sugar chainshave variable size, even at a single glycosyla-tion site. One way around this problem is toget rid of the sugar chains altogether andtag the peptides that once had the sugarchains. These papers do just that.

In both reports3,4, the researchers firstimmobilize the sugar chain containing pro-teins/peptides and then selectively releasethe peptides with peptide-N-glycosidase F(PNGase F) for analysis. Tagging thereleased peptides with heavy isotopesassures that only formerly glycosylated pep-

Mass spectrometry provides sweetinspirationHudson Freeze

Two new methods facilitate the profiling of N-glycosylated proteins incomplex mixtures.

The author is at The Burnham Institute,10901 North Torrey Pines Road, La Jolla,CA 92037, USA.e-mail: (hudson@burnham.org)

directed mutations in mice and rats is a mat-ter of ordering an ES cell or sperm sample,and the focus can be on the biology of thephenotypes produced.

1. Zan, Y. et al. Nat. Biotechnol. 21, 645–651(2003).

2. Valenzuela, D.M. et al. Nat. Biotechnol. 21,652–659 (2003).

3. Coghill, E.L. et al. Nat. Genet. 30, 255–256(2002).

4. Chen, Y., Schimenti, J. & Magnuson, T. Mamm.Genome 11, 598–602 (2000).

5. Stanford, W.L., Cohn, J.B. & Cordes, S.P. Nat. Rev.Genet. 2, 756–768 (2001).

6. Nagy, A. Genesis 26, 99–109 (2000).7. Eggan, K. et al. Proc. Natl. Acad. Sci. USA 98,

6209–6214 (2001).

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tides pass the final inspection (Fig.1).In the first paper, Zhang et al.3 exploit

the fact that glycans have at least one set of cis hydroxyl groups, and they use perio-date to oxidize them to aldehydes. Thesereact with hydrazide groups on a solid sup-port forming covalent hydrazone bonds.Nonglycosylated proteins do not react andare washed away. The immobilized glyco-proteins are then digested with trypsin to remove nonlinked peptides. Finally,N-glycan-specific PNGase F digestion freesthe formerly N-glycosylated peptides fromtheir sugar bondage. As the enzyme is a gly-coamidase, releasing the peptide conve-niently converts asparagine to aspartic acid,while retaining the sugar chain on the bead.The highly purified peptides are separated

N E W S A N D V I E W S

and fingerprinted by microcapillary high-performance liquid chromatography elec-trospray ionization (µLC-ESI) tandem MS or µLC matrix-assisted laser desorp-tion/ionization (MALDI) tandem MS toidentify the protein. When quantificationor a comparison of two samples is required,prior to PNGase F treatment, the α-aminogroups of the immobilized glycopeptidecan be labeled with light or heavy succinicanhydride to introduce a four-unit massdifference.

Zhang et al.3 first validate their methodon human serum. They identify 145 gly-copeptides from 57 unique proteins, whichaccounts for the great majority of previ-ously identified glycoproteins and knownoccupied glycosylation sites. The analysis

time is fast, and protein size, abundance,isoelectric points (pIs) and sugar chaincomplexity do not bias recovery. A humanserum sample can be analyzed in 2 h ofµLC-ESI-tandem MS time, and although asingle run may not detect all known species,altering the proteolytic enzyme or massrange scan can generate and detect differentpeptides. Comparison of isotopicallytagged samples can determine whether aglycosylation site is occupied under differ-ent physiological states or in such diseasesas CDG.

When the method is recruited to analyzecell-surface proteins using crude mem-brane samples, it finds many of theexpected members, but it is difficult toknow how many glycoproteins remainedundetected. Those missing might containperiodate-insensitive sugar chains, butmore likely, PNGase F did not cleave them.For instance, fucose α 1,3-N-acetylglu-cosamine in the core region of the glycansblocks cleavage. Fortunately, anotherenzyme, PNGase A, can cleave these chains.However, other resistant species may occurin mammalian cells. PNGase F is the besttool available, but there is no guarantee thatit cleaves all N-linked chains.

Can this method be used to locate pro-teins with carbohydrates linked to serine or threonine residues (O-linked chains)?The periodate oxidation will work, butunfortunately, there is no PNGase F equiva-lent for O-linked oligosaccharides.However, β-elimination of O-linkedoligosaccharides, including O-mannose7-and O-fucose8-linked forms produces anunsaturated bond on the peptide that couldbe tagged.

In the second paper, Kaji et al.4 focus onidentifying N-glycosylated proteins inCaenorhabditis elegans. They first purifyglycoproteins on concanavalin A beads,which selectively bind glycoproteins withhigh-mannose and hybrid-type N-glycans.Trypsin digestion generates glycopeptides,which are isolated once again on con-canavalin A beads. Eluted glycopeptides arereleased by PNGase F cleavage in the pres-ence of H2

18O. This treatment generates18O aspartic acid–labeled peptides, whichare fractionated by multi-dimensional liq-uid chromatography and identified by MSanalysis. They identify 400 N-glycosylationsites on 250 proteins, and determine fre-quency of ‘X’ in the second position of theconsensus sequence. Significantly, they findfour sites containing cysteine at the thirdposition, rather than the expected Thr(278/400) and Ser (118/400).

628 VOLUME 21 NUMBER 6 JUNE 2003 NATURE BIOTECHNOLOGY

Figure 1 Identification of N-linkedglycoproteins from complexmixtures. The method of Zhang etal. (left side) oxidizes hydroxylgroups on sugar chains to aldehydesusing periodate. Peptides are notmodified, but any glycoprotein cannow be covalently bound to beadscontaining a hydrazide. Washingremoves unbound proteins, and thetrapped proteins are digested withtrypsin. Another wash removesdigested unbound peptidefragments. The immobilizedglycopeptides are next digested witha glycoamidase, PNGase F, whichcleaves the N-acetylglucosamine-asparagine amide bond. Thisconverts asparagine to aspartic acidreleasing the peptide and retainingthe sugar chain on the bead.Peptides are separated andidentified by LC-MS. In the methodof Kaji et al. (right side), theglycoproteins are first bound to alectin column—in this case, thelectin is concanavalin A—and theneluted with a sugar. The highlyenriched glycoproteins are thentrypsin-digested in solution and theglycopeptides isolated again on thelectin column. After elution, thepeptides are concentrated anddigested with PNGase F in H2

18O,which generates an 18O-Asp-taggedpeptide. The sugar chain alsobecomes 18O-tagged when theamino group is hydrolyzed. The 18O-Asp-tagged peptides are thenseparated and identified.

Blow-up of linkage

Protein

Glycoprotein

Generic sugar chain

Bead

Proteins and glycoproteins

Oxidizesugarchain

Couple to hydrazidebead

Zhang et al.

Purify glycoproteinson con A beads

O

Purify glycopeptideson con A beadsand elute

–X–X–Asp–X–Thr/Ser–X–

X–Asp–X–Thr/Ser

X–Asn–X–Thr/Ser

Linkage region afterPNGase F digestion

Generic linkage region

–X–X–Asp–X–Thr/Ser–X–

Analysis/identification

PNGase Fdigest in solution(H2

18O)

PNGase Fdigest,releasepeptide

Trypsin digestpurifiedglycoproteins

Trypsin digeston bead

Kaji et al.

18O

18O

NH

NH

NH

NH

NH

NH

X–Asn–X–Thr/Ser

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carbohydrate-dependent binding of P-selectinglycoprotein ligand-1 to P-selectin10. Thereal ligand is a fucosylated sugar chainembedded in a patch of modified aminoacids. The sensitive tools are now at hand toinvestigate many important questionsregarding the glycosylation status of com-plex protein samples.

1. Varki, A. Glycobiology 3, 97–130 (1993).2. Freeze, H. Glycobiology 11, 129–143 (2001).

3. Zhang, H. et al., Nat. Biotechnol. 21, 660–666(2003).

4. Kaji, H. et al., Nat. Biotechnol. 21, 667–672 (2003).5. Nishikawa A. & Mizuno S. Biochem. J. 355, 245–248

(2001).6. Apweiler, R., Hermjakob, H. & Sharon, N. Biochim.

Biophys. Acta 1473, 4–8 (1999).7. Martin, P. & Freeze, H. Glycobiology, in press (2003). 8. Haltiwanger, R. Curr. Opin. Struct. Biol. 12, 593–598

(2002).9. Kasturi, L., Chen, H. & Shakin-Eshleman, S.H.

Biochem. J. 323, 415–419 (1997).10. Leppanen, A. et al. J. Biol. Chem. 275,

39569–39578 (2000).

NATURE BIOTECHNOLOGY VOLUME 21 NUMBER 6 JUNE 2003 629

An advantage of this method is that different lectins can be used to capture proteins with selected glycans; moreover,the noncovalently bound sugar chains can be eluted with appropriate monosac-charides for analysis. Even though hydroly-sis of the PNGase F–generated glyco-sylamine also tags the free oligosaccharideswith 18O, they cannot be traced to their par-ent peptides.

The approach does have several limitations.First, using a lectin column to select glycopep-tides only binds a subset of N-glycans.Most known C. elegans glycoproteins areexpected to bind, but this is not true forthose from more complex organisms.Peptides derived from very abundant pro-teins (e.g., histones) will also contaminatethe bound glycopeptides. C. elegans also con-tains PNGase F–resistant N-glycans, so thesewill not be counted. This may be a more sig-nificant problem in the analysis of glycosyla-tion in lower organisms where N-glycanstructure, and thus PNGase F–resistance, ismostly unexplored. 18O-tagging during thecleavage is important to avoid false posi-tives because nonenzymatic deamidation ofasparagine to aspartic acid does occur andis indistinguishable from the PNGaseF–catalyzed reaction. Careful comparisonsof 16O and 18O-labeled peptides can bedone by high-resolution MALDI-MS toaddress this issue, but for some laboratoriesthe cost of H2

18O may be prohibitive.Notwithstanding their limitations, these

methods should make it possible for thefirst time to analyze complex protein mix-tures to determine whether N-glycosylationsites of specific proteins are equally occu-pied under different physiological states orin different tissues. For instance, site occu-pancy in DNase I varies, and perhaps this istrue of many proteins. Asn-Asp-Ser andAsn-Glu-Ser sequences are thought to beinefficiently N-glycosylated9. Criticalreceptors such as glycine receptor, sodiumchannel protein, glutamate receptor 3,NMDAR1, metabotropic glutamate recep-tor 1 beta, neurexin 4, brevican, T-cellreceptor beta chain and protocadheringamma all contain such sequences.Although clearly speculation at this point,variable glycosylation might influence lig-and binding or formation of signaling com-plexes in the membrane and this now canbe investigated. Sugar and peptide special-ists alike can exploit the methods and arelikely to find that the peptides surroundingsome glycosylation sites have additional mod-ifications. For instance, multiple tyrosine-sul-fate residues are required for efficient

In recent years, we have witnessed the discov-ery of a major and universal mechanism ofgene regulation called RNA silencing or RNAinterference (RNAi). Although the mecha-nism by which RNAi controls gene regulationis only partly understood (Fig. 1a), consider-able interest is focusing on the putative thera-peutic applications of these molecules,particularly for fighting viral infections. Inthis issue, McCaffrey et al.1 demonstrate thatshort hairpin RNAs (shRNAs) block replica-tion of hepatitis B virus (HBV) in vivo, bring-ing us one step closer to the use of RNAi as anantiviral therapy.

The idea that RNAi could be applied inantiviral therapies was first suggested bystudies in plants, where RNAi appears tofunction as a major natural antiviral mech-anism. Several lines of evidence supportthis contention: infection by plant viruseselicits strong gene silencing; plant virusesencode a variety of inhibitors of the RNAsilencing machinery; and mutations ingenes that encode for the RNA silencingmachinery result in enhanced susceptibilityto virus infection2,3. Viral replication canalso be efficiently suppressed by experi-mentally induced RNA silencing. In animalcells, initial experiments first focused onthe simple issue of whether mammalianviruses are susceptible to RNAi or not.Although any RNA can potentially be sub-ject to degradation by the RNAi machinery,it was not clear whether viral RNAs would

also be effectively targeted. Virus genomesare often protected by a proteinaceousstructure or through their association withcellular membranes during replication;although many different types of viruseshave been shown to be susceptible to RNAiin tissue culture, whether it would be possi-ble to induce an effective antiviral RNAiresponse in vivo in the context of multiplesystemic regulatory systems, such as theimmune system, was not known.

McCaffrey et al. employ a techniquecalled hydrodynamic transfection to intro-duce plasmids encoding the HBV genometogether with HBV-specific shRNAs intomouse liver. This technique allows efficientgene transfer by rapidly injecting a largevolume of DNA solution into mice via thetail vein (Fig. 1b). Among the organs trans-fected with the exogenous HBV DNA (liver,spleen, kidney and pancreas), the livershowed the highest levels of gene expressionwith approximately 40% of hepatocytesexpressing the transgenes. The authorsshow that the HBV-specific shRNAs signifi-cantly reduce viral mRNAs and proteinexpression, thus inhibiting virus replicationin hepatocytes of HB-infected mice. Theseresults, obtained using transient in vivocotransfection of plasmids, provide animportant proof of principle that antiviralactivity by RNAi can be achieved in animals.They also open the door for what promisesto be a very exciting and prolific new field ofantiviral therapeutics.

An important question to be addressed iswhether or not there is a functional interac-tion between the RNAi machinery and themammalian immune system. This reportcompares the efficiency of RNA silencing in

The author is in the Department of Microbiologyand Immunology, Box 2280, University ofCalifornia, San Francisco, CA 94143-2280, USA.e-mail: (andino@cgl.ucsf.edu)

RNAi puts a lid on virus replicationRaul Andino

Effective inhibition of hepatitis B virus replication in mice by RNAinterference suggests an antiviral treatment strategy.

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