fractions (7) and experiments of Ratnam et al. (8) all indicate that certain regions between M3 and M4, and the C termini

Transcription

1 Proc. Natl. Acad. Sc. USA Vol. 86, pp , September 1989 Cell Bology Structure, olgosaccharde structures, and posttranslatonally modfed stes of the ncotnc acetylcholne receptor (glycoylatlon/lon channels/lqud secondary on m- spemetry/massspectrometry/multmhanne array detecton) L. POULTER*t*, J. P. EARNEST, R. M. STROUD*, AND A. L. BURLINGAME*t Departments of *Pharmaceutcal Chemstry and Bochemstry and Bophyscs, and the tmass Spectrometry Faclty, Unversty of Calforna, San Francsco, CA Communcated by Manuel F. Morales, June 19, 1989 ABSTRACT Usng mass spectrometry, we have examned the transmembrane topography of the ncotnc acetylcholne receptor, a fve-subunt glycosylated proten complex that forms a gated on channel n the neuromuscular juncton. The prmary sequences of the four polypeptde chans makng up the acetylcholne receptor from Torpedo calfornca contan many possble stes for glycosylaton or phosphorylaton. We have used lqud secondary on mass spectrometry to dentfy posttranslatonally modfed resdues and to determne the ntact olgosaccharde structures of the carbohydrate present on the acetylcholne receptor. Asparagne-143 of the a subunt (n consensus numberng) s shown to be glycosylated wth hgh-mannose olgosaccharde. Asparagne-453 of the y subunt s not glycosylated, a fact that bears on the queston of the orentatons of putatve transmembranous helces M3, MA, and M4. The structures of the sx major acetylcholne receptor olgosacchardes are determned: the major components (70%) are of the hgh-mannose type, wth b-, tr-, and tetraantennary complex olgosacchardes makng up =22 mol% of the total carbohydrate. Ths applcaton of a multchannel array detector mass spectrometer provded a breakthrough n senstvty that allowed us to dentfy the ste of attachment of, and the sequence of, olgosacchardes on a 300-kDa membrane proten from only 5 pmol of the solated olgosaccharde. How the ncotnc acetylcholne receptor (AcChoR) medates on conductance n response to bndng acetylcholne at the neuromuscular juncton s of crtcal mportance to understandng the functon of the famly of related neuroreceptors (1). The molecular structure of the AcChoR contans fve smlar subunts that surround the on-conductng channel, shown to le n the center (2, 3). Whle the prmary structures of all four subunt types of the acetylcholne receptor from Torpedo calfornca are homologous from N to C termn (4), there s as yet no dentfcaton of all regons that are transmembranous. However, n each of the fve subunts (a2zy8), there are four hydrophobc stretches of amno acds that are presumed to le across the membrane (5). Ste-drected mutageness shows that resdues of the second helx, M2, determne aspects of the conductvty (6). On the bass of our x-ray dffracton evdence for orented a-helces perpendcular to the membrane we proposed that the on channel could be formed wthn a bundle of such a-helces. More polar surfaces could provde the lnng of the pore. A ffth amphpathc a-helx, whch may also be transmembranous (MA), s apparent from the AcChoR prmary sequence. Our hypothess that these helces could also contrbute to the pore led to experments desgned to determne the peptde topography, especally n the regon of MA, M4, and the C termnus. Our electron mcroscopc localzaton of peptdespecfc antbodes on ntact natve tssue and on subcellular The publcaton costs of ths artcle were defrayed n part by page charge payment. Ths artcle must therefore be hereby marked "advertsement" n accordance wth 18 U.S.C solely to ndcate ths fact. fractons (7) and experments of Ratnam et al. (8) all ndcate that certan regons between M3 and M4, and the C termn are cytoplasmc. However, related sequence famles n the y-amnobutyrc acd and glycne receptors, whch clearly seem to have smlar structure to the AcChoR, do not contan an amphpathc helx lke MA (9). Ths conjecture remans the strongest evdence that MA does not cross the membrane and so cannot form part of the channel. We desgned a strategy that n prncple can determne complete topography of any such membrane proten; we begn by reportng ts applcaton to unambguous defnton of posttranslatonally modfed stes that contan topographcal nformaton. On the common presumpton that glycosylated stes n membrane protens are extracellular, we sought to determne where these are located. One consensus ste for N-glycosylaton (Asn-Xaa-Ser/ Thr) les n the regon between MA and M4 n the y chan alone; glycosylaton here, had t occurred, would serve to defne topography n ths crtcal regon. We show here that ths ste does not carry polysaccharde, agan leavng open the absolute determnaton of sdedness of the MA-M4 lnkng regon. We also fnd that Asn-143ac II s glycosylated wth hgh-mannose olgosaccharde, and we elucdate the structures of >90%o of the olgosacchardes attached to the AcChoR. The methods we descrbe now provde a means of unambguous resoluton of AcChoR topography by usng exogenous labels from wthn the membrane as well as from ether sde of the membrane. MATERIALS AND METHODS AcChoR Purfcaton. AcChoR was affnty purfed from T. calfornca accordng to ref. 10 wth mnor changes: NaF (100 mm) was substtuted for NaCl and protease nhbtors (20 ;kg of leupeptn per ml, 20 ug of antpan per ml, 20 unts of trasylol per ml) were ncluded to nhbt endogenous phosphorylases. In preparng the acetylcholne affnty column, AffGel-10 dervatzed wth cystamne was used nstead of AffGel-401. Resdual carbamoylcholne and cholate were removed by dalyss, and then the dalyss buffer was exchanged for NH4HCO3 (50 mm; ph 7.8). AcChoR was reduced wth dthothretol (10 mm) n NH4HCO3 (50 mm; ph ) under N2 for 2 hr at 370C, the thols were alkylated wth 20 mm odoacetate at room temperature for 1 hr. and then reagents were removed by dalyss aganst ammonum bcarbonate (50 mm; ph 7.8). Abbrevatons: AcChoR, acetylcholne receptor; ABEE, p-amnobenzoc acd ethyl ester; LSIMS, lqud secondary on mass spectrometry; PNGase F, peptde N-glycosdase. tpresent address: Department of Botechnology, ICI Pharmaceutcals, Meresde, Alderley Park, Macclesfeld, Cheshre SK10 4TG, U.K. 1To whom reprnt requests should be addressed. "Resdues wth the subunt and "C" subscrpt refer to the consensus numberng scheme of Stroud and Fner-Moore (2); resdues wth only the subunt subscrpt are sequental n the prmary sequence. 6645

2 6,6 Cell Bology: Poulter et al. Fractonaton of AcChoR for Mass Spectrometry. Tryptc and Staphylococcus aureus V8 dgestons ofthe receptor (500 Ag; 2 nmol) were carred out n ammonum bcarbonate (50 mm; ph 7.8) wth 0.1% NaDodSO4. After dgeston, NaDodSO4 was removed by precptaton wth guandne hydrochlorde (11). Peptdes and glycopeptdes were separated by gel fltraton on a G-50 column usng 50 mm ammonum bcarbonate as eluent and montorng at 210 and 280 nm. Alquots of these fractons were acd hydrolyzed to release monosacchardes, whch were then dervatzed as trmethylslyl methyl glycosdes. Gas chromatographc analyss of the dervatves gave the monosaccharde composton and therefore ndcated whether the olgosacchardes were of the hgh-mannose or complex types. Alquots of the olgosaccharde-contanng fractons were treated wth peptde N- glycosdase F (PNGase F), whch cleaves hgh-mannose, hybrd, and complex glycans from N-lnked glycosylaton stes, whle convertng asparagne to aspartc acd (12). Reverse-phase HPLC [on a Vydac column (C18, C4, and bphenyl), usng water/0.1% trfluoroacetc acd and acetontrle/0.09%o trfluoroacetc acd as the aqueous and organc phases, respectvely] of peptdes before and after deglycosylaton further fractonated the peptdes for analyss by mass spectrometry. Olgosacchardes were released from N-lnked glycosylaton stes by dgeston of AcChoR (500,ug; 2 nmol) wth PNGase F. In a modfcaton of prevously descrbed methods (13, 14), AcChoR was dgested n ammonum bcarbonate buffer (100 mm; ph 8.6) wth 0.1% NaDodSO4 and 0.6% Nondet P-40 at 37 C for 18 hr. Analytcal NaDodSO4/PAGE (15) showed dgeston to be complete. NaDodSO4 was precptated as descrbed earler, and olgosacchardes were separated from proten and resdual detergent by passage through a dsposable reverse-phase matrx (Sep-Pak C18 cartrdge; Waters). The aqueous fracton, contanng all released olgosacchardes, was ether lyophlzed drectly or treated wth neuramndase before lyophlzaton, and then dervatzed accordng to a modfcaton of the method descrbed by Wang et al. (16) n whch p-amnobenzoc acd ethyl ester (ABEE) (16.5 mg) and sodum cyanoborohydrde (3.5 mg) were added to the sample n 100 ul of methanol/ acetc acd (9:1, vol/vol), and the soluton was ncubated at 80 C for 40 mn. After coolng, 500,ul of chloroform was added and the mxture was extracted twce wth water (500,ul). The combned water layers were lyophlzed and the dervatzed olgosacchardes were purfed by HPLC. An amnopropyl column was used for desalylated olgosacchardes, wth a gradent of 80-40%o acetontrle over 40 mn, whle montorng at 320 nm. Salylated structures were purfed on C18 or C4 reverse-phase columns usng water and acetontrle as the aqueous and organc phases and ncorporatng the followng eluton program: 0-10 mn, 100% water; mn, 0-60% acetontrle. The eluent was montored at 320 nm. Mass Spectrometry. All peptde fractons collected from each reverse-phase HPLC analyss were subjected to lqud secondary on mass spectrometry (LSIMS) n the postve on mode (14). Expermentally derved molecular weghts, together wth sequences obtaned from peptde fragmentaton, were used to assgn peptdes to regons wthn one of the four receptor subunts. The expected masses of peptdes produced by proteolytc cleavage were calculated from the cdna sequence (4). Mass spectra of dervatzed olgosacchardes were recorded n the postve-on mode to obtan molecular weghts and purty. In ths mode, fragmentaton of ths dervatve s mnmal, allowng dentfcaton of the molecular on at hgh senstvty (14). Negatve-on spectra were recorded to obtan fragmentaton and to determne olgosaccharde sequences (14). Proc. Natl. Acad. Sc. USA 86 (1989) All mass spectra of peptdes and olgosacchardes, unless otherwse stated, were recorded wth a Kratos MS 50-S double-focusng mass spectrometer, equpped wth a hghfeld magnet, a LSIMS source (17, 18), and a postacceleraton detector (10 kev). In poneerng experments n whch multchannel array detecton was used, negatve on spectra were recorded wth a Kratos Concept. The characterstcs of the multchannel electrooptcal array detecton system used n these studes have been reported (19). Negatve-on LSIMS spectra were obtaned by computer-controlled acquston from the array detector n sequental 4% mass segments (20). RESULTS Peptde Mappng of AcChoR Subunts. AcChoR peptdes dentfed by mass spectrometry are lsted n Fg. 1. The regons of the AcChoR that were mapped by mass spectrometry are heavy-lned on the predcted secondary structure dagram of the consensus subunt n Fg. 1. The N-termnal peptdes, the cytoplasmc loops downstream from M3, and the proposed amphpathc helcal domans (MA) were most readly dgested and mapped by mass spectrometry, reflectng an ncreased number of trypsn/v8 cleavage stes n these regons. Resdues and the resdues toward the C termnus from 490 dd have cleavage stes but were generally resstant to cleavage. The hydrophobc putatve transmembrane domans lack V8 and trypsn cleavage stes and are thus not yet amenable to mass spectrometry analyss. A reverse-phase HPLC chromatogram of one of the peptde fractons clearly showed an absence of one peak upon deglycosylaton wth PNGase F (data not shown). One of the peptdes n the peak fracton after deglycosylaton had a molecular weght MH' = 1980 (nomnal mass), whch was not present n the mass spectrum of ths HPLC fracton pror to deglycosylaton (Fg. 2). Ths molecular weght corresponds to the peptde Ile-Ile-Val-Thr-Hs-Phe-Pro-Phe- Asp-Gln-Gln-Asp-Cys-Thr-Met-Lys, whch s the expected product of PNGase F dgeston of the glycosylated peptde ac; a. Ths demonstrates that Asn-143ac s glycosylated n the natve state. Moreover, GC analyss ofthe trmethylslyl methyl glycosdes of monosacchardes released from ths glycopeptde demonstrated that only mannose and N-acetylglucosamne were present, ndcatng that a hgh-mannose structure s attached at ths ste. Another potental glycosylaton ste that was mapped by mass spectrometry was Asn-453,yc (431y). A molecular on wth MH+ = 1181 was found, the mass of whch matches an unglycosylated peptde from the 'y subunt, Ser-Thr-Lys- Glu-Gln-Asn-Asp-Ser-Hs-Ser-Glu. The equvalent peptde wth aspartate substtuted for asparagne was not found n any PNGase-treated sample, demonstratng that the y subunt s not glycosylated at Asn453yC. All of the seven putatve stes for phosphorylaton by camp-dependent proten knase, proten knase C, or tyrosne knase were mapped n these experments, and no peptdes contanng phosphoserne, phosphotyrosne, or phosphothreonne were detected. Olgosaccharde Analyss. Mass spectrometrc analyses showed the major fracton of receptor olgosacchardes (=70%o as judged by ntegrated absorbance at 320 nm durng HPLC purfcatons) to be hgh-mannose structures wth compostons Man8GlcNAc2-ABEE and Man9GlcNAc2- ABEE, as judged by postve-on mass spectra, whch showed protonated molecular ons at MH+ = 1870 and MH+ = 2032, respectvely. Full detals of the fragmentaton observed are provded n Fg. 3, whch shows the negatve-on mass spectrum recorded usng =10 ng (5 pmol) of Man8GlcNAc2-ABEE usng computer-controlled mass wndow steppng and multchannel array detecton (20).

3 Cell Bology: Poulter et al. A a4 Thr5 -Glul3 Leu56 -Arg64 Trp67 -Lys76 1le116-Lysl25 Ile130-Lysl55 Serl62-Glul72 Ile315-Lys38O Leu430-Gly437 'Y E. LyslO -Argl Gly74 -Arg79 Vall56-Glul66 Asnl8l-Lys20l Lys346-Arg38l Asp405-Glu jc p.0 PC Serl -Glu Serl26-Lysl3l Vall56-Argl Glu172-Lysl9l Ser308-Arg Ala351-Lys Phe421-Lys oc Leu7 -Lys25 Leu278-Lys290 Thr316-Lys327 Ile337-Arg342 Gln348-Arg395 Ile400-Glu407 Ser421-Lys B Proc. Natl. Acad. Sc. USA 86 (1989) 6647 FIG. 1. AcChoR peptdes dentfed by LSIMS. (A) Mapped peptdes of each subunt are dentfed by both prmary sequence numberng and consensus numberng (2). (B) The consensus subunt secondary structure model s used to llustrate the locatons of the dentfed peptdes. Subunt-specfc cleavage stes are shown: *, a chan; A,, chan; *, y chan; o, 8 chan. Putatve glycosylaton stes are denoted by stars, accompaned by the subunt symbol where the stes are not conserved n all subunts. Some of the conserved amno acds are ncluded as reference ponts; they are denoted by the sngle-letter abbrevaton and the consensus number. Analyss of neuramndase-treated samples showed the major speces present n the remanng 30o of olgosaccharde to be nonfucosylated tr-, tetra-, and bantennary complex structures, wth MH+ = 2155, MH+ = 2520, and MH+ = 1790, respectvely. A fourth component wth MH+ = 2358 had the composton Man3Gal3GlcNAc6 as judged by molecular weght and monosaccharde analyss. Fragmentaton observed n the negatve-on mode ndcated a tr-antennary structure wth a bsectng N-acetylglucosamne resdue. All structures observed by mass spectrometry are detaled n Fg. 4. In our earler experments, salc acd resdues were removed from all olgosacchardes by neuramndase treatment to smplfy olgosaccharde analyss. More recently, we have prepared and purfed salo-abee dervatves from receptor olgosacchardes. Fg. 5 shows a spectrum obtaned by usng 1,ug of an ABEE-dervatzed trsalo-trantennary structure purfed by reverse-phase HPLC. The senstvty acheved wth ths dervatzaton s roughly 10- to 20-fold greater than that attaned n the analyss of undervatzed salo-olgosacchardes and s due to the ntroducton of the hydrophobc group, whch ncreases the surface actvty of the olgosaccharde n the lqud matrx durng mass spectrometry analyss (21). The eluton postons of olgosacchardes n the reverse phase HPLC chromatograms of salo-abee structures ndcate that all of the complex olgosacchardes are hghly salylated. DISCUSSION Glycosylaton. We have determned the structures of the major olgosacchardes n AcChoR from T. calfornca; two are of the hgh-mannose type, and four are complex structures. Hgh-mannose olgosacchardes are attached to every a chan at Asn-143ac (141J). All four subunts were shown to be glycosylated (22) by olgosacchardes of ether the hghmannose or complex type. Nomoto et al. (23) demonstrated that hgh-mannose olgosacchardes are present on all subunts, wth complex olgosacchardes present only on the y and 8 chans. No olgosaccharde structures contanng fucose were found by mass spectrometry. Ths fndng s n contrast to the results of Nomoto et al. (23), who ndcate fucosylated chtobose core on a proporton of b-, tr-, and tetraantennary structures. Ther evdence, however, s prmarly based on a comparson of HPLC retenton tmes wth those of putatve standards, whch clearly s an nadequate sole crteron for olgosaccharde dentfcaton. Prmary sequences of the four subunts reveal one possble ste for N-lnked glycosylaton (consensus sequence s Asn- Xaa-Ser/Thr) on a, one on 13, four on y, and three on 8, as shown n Fg. 1. Because the a chan s glycosylated, and because the prmary sequence shows only one ste for N-glycosylaton, ths ste was assumed to be the pont of carbohydrate attachment. Nevertheless, the postve dentfcaton of glycosylated Asn-143ac by mass spectrometry s Untreated salmle MH + Assanment IFADDIDISDISDISGK (cx) IIVTHFPFDQQNCTMK 1611 VIFQTPLIKNPDVK (a) 1750 NKIFADDIDISDISGK (a) \ PNGase F 2022 IFADDIDISDISGKQVTGE (ax) 1980 IIVTHFPFDQQDCTMK (a) IIVTHFPFDQODCTMK 1611 I _ - I PNGase F treated sample C m/z FIG. 2. The postve-on LSIMS spectrum of the peptde fracton contanng glycosylaton ste Asn-143,tc before (Upper) and after (Lower) PNGase F dgeston. The molecular on appearng at m/z 1980 (nomnal mass) corresponds exactly to a peptde contanng Asn-143,ac, wth the converson of Asn-143ac to aspartc acd after deglycosylaton (see Inset). Amno acds are denoted by the sngle-letter code. (Lower) A poorer sgnal to nose rato s observed and s probably due to traces of detergent that were ncluded n the enzymatc dgeston. Other molecular ons, observed at m/z 1508, 1611, 1750, and 2022, correspond to peptdes from the a subunt, and ther denttes are also provded.

4 6648 Cell Bology: Poulter et al. Proc. Natl. Acad. Sc. USA 86 (1989) 100I (M - H) 40! l 100 0o Nel 4H ' " FIG. 3. The negatve-on LSIMS spectrum ' obtaned on 10 ng (5 pmol) of Man8GlcNAc2- ABEE from AcChoR, usng multchannel array detecton n combnaton wth computer-con- Man Mange 177>058 trolled mass wndow steppng. Losses of one, Man-Manl Man-Marn-Man two, three, and four mannose unts are prom- Man-GIcNAc-GIcNAc-ABEE Man-GcNAc-GIcNAc-ABEE nent, whch s n agreement wth the structure Man-Man-Man Man-Man predcted by the 1H NMR work of Nomoto et al (23). A weak fragment on, correspondng to the loss of Man5, s also detected well above the 20 LI J., I A.. I Ik.- N-142,60 L nl kl..it li L L. AW nose level (ons marked by astersks arse from *" 1058A40 protonated glycerol matrx cluster ons, n d \ whch the sample was dssolved; ref. 16); ths e ll I 1 6 f on could arse from two bond cleavages or from I the presence of a mnor component of somerc.._. _._._ structure. No mnor component was detected by 'H NMR or wthout the use of the array system. the frst nstance of assgnng a glycosylaton ste to a specfc amno acd on the acetylcholne receptor. It has been postulated that dfferental glycosylaton of the two a chans s responsble for what may be dfferent affntes of compettve antagonsts for the receptor (24, 25). Ths postulate was supported by peptde mappng experments n ANOACCHARIDE STRUCTUREE M - Ma Man GlcNAc 9 2 Gal3 Man3 GIcNAc5 Gal Man GlcNAc Gal 2Man3 GIcNAc4 Gal4 Man3 GlcNAc6 Man8 GIcNAc *3M- G-Gal-2 al-2 M-M-M M-M \ M-M-Mx,M G-G - s M-M *g-g G g-g-m NA-G 9 -G-M-' M- G-G- 9- G-M MG-G- 9-G I GLECuAR WEIGHT APPROXIMATE MOLE% FIG. 4. The molecular weghts, monosaccharde composton, structures of olgosacchardes (as deduced by fragmentaton analyss), and approxmate molar percentages of the major speces are recorded. M, mannose; G, N-acetylglucosamne; g, galactose. The molecular weghts shown reflect the olgosaccharde mnus the ABEE group (Mr, 149). Approxmate mol % values were calculated from ntegrated peak szes from the HPLC chromatograms. The lnkage Mal-6M as opposed to Mal-2M was shown by methylaton analyss and s n accordance wth the 1H NMR data of Nomoto et al. (23). The bantennary branch of the trantennary structures (astersk) s shown attached to the Mal-6M branch of the olgosaccharde. Ths nformaton cannot be nferred from fragmentaton data but was chosen to be n accordance wth the HPLC data of Nomoto et al. (23). 2 whch two V8 fragments of the a chan, an 18-kDa fragment and a 20-kDa fragment, were both beleved to contan Asn- 143Cc, yet only one was glycosylated (26). However, Pedersen et al. (27) demonstrated that under certan condtons the 20-kDa V8 fragment s composed of two dstnct 20-kDa fragments, the major component beng Ser-173-Glu-335 (contanng no glycosylaton ste), and the mnor component beng glycosylated Val-46-Glu-172. Our results demonstrate that all a chans have carbohydrate attached, snce otherwse a nonglycosylated a-peptde contanng Asn-143ac would have been detected by mass spectrometry even f t were a mnor component. The possblty stll remans that there could be mnor dfferences n the a-chan carbohydrates; n the future ths wll be evdent from mass spectrometrc analyss and quantfcaton of olgosacchardes released from the purfed a subunts. There are four consensus stes for glycosylaton on the y chan. The four asparagnes are (n consensus numberng): NANA -VGl-~GIcNAc 2370 NANA- Gal - GIcNAc-Man 1552 NANA- Gal-GIcNAc Man Man-GIcNAc-GIcNAc-ABEE ' -A-,w A - LLu. (M-H) , (M-2H+Na) 155l... : m/z ^I~.L s_ L,119.1a._.2. -m/z &-.M6 HL AL..- PhIlk,...j lw-.00%mllawlall.pwoow -. _ ".k- A A -' FIG. 5. The negatve-on LSIMS spectrum of a trantennary salo-olgosaccharde dervatzed wth the ABEE reagent, whch was obtaned on '300 pmol (1 gg) of materal. Fragmentaton of glycosdc bonds s observed wth charge retenton at the dervatzed reducng termnus. *, Speces result from glycosdc bond cleavage and the loss of a water molecule from the parent molecular on; #, speces nclude C1 of the non-reducng termnal monosaccharde.

5 Cell Bology: Poulter et al. and 143, whch le n the soluble doman N termnal to Ml; 315, whch accordng to secondary structure predctons s located on the cytoplasmc sde of M2; and 453, between MA and M4 (Fg. 1). We have establshed that Asn-453.yc s not glycosylated, demonstratng that there are a maxmum of three olgosacchardes attached to the y chan. If the y chan does carry three olgosacchardes, as demonstrated by Anderson and Blobel (28), one of the olgosacchardes must be attached at Asn-316; ths would mply that the orentaton of M3 n the blayer has to be nverted. The drect sequencng and determnaton of the branched structure from fragmentaton of the ABEE-dervatzed olgosacchardes mples that monoclonal antbodes drected aganst specfc olgosacchardes can now be made and used as probes for membrane protens present n purfed form at the pcomolar level, to the femtomolar level n the future. It has not escaped our notce that ths may be useful n localzng specfc rarely expressed membrane protens. AcChoR Phosphorylaton. Three endogenous proten knases have been shown to phosphorylate specfc subunts of the Torpedo acetylcholne receptor; namely, camp-dependent proten knase, proten knase C, and a tyrosne-specfc proten knase (revewed n ref. 29). All of the seven possble stes for phosphorylaton by these three endogenous proten knases were located and examned n ths work, and no phosphorylated peptdes were detected. It s possble that these stes are lable to endogenous phosphatases, even n the contnued presence of sodum fluorde and protease nhbtors. Advances n Structure Determnaton Usng Mass Spectrometry. Ths study of chemcal and posttranslatonal modfcaton of a complex multsubunt membrane proten was carred out on a total of 4 nmol of sample (1 mg of AcChoR). A combnaton of the protocol descrbed here and the senstvty of hgh-performance tandem sector systems wth electrooptcal multchannel array detecton wll permt sequencng of the proten, structural characterzaton of the posttranslatonal modfcatons, and determnaton of the nature and stes of covalent modfcatons resultng from photoaffnty experments at the subnanomole level (20). The mass spectrometrc analyses of olgosacchardes demonstrate that the ABEE dervatve s superor to the undervatzed olgosaccharde, snce the ntroducton of a hydrophobc group greatly ncreases the surface actvty of the compound and hence the senstvty of the analyses (21). In addton, excellent fragmentaton of the olgosacchardes, predomnantly at successve glycosdc lnkages, s provded n the negatve-on mode (13, 14). Also, ths dervatzaton adds only 149 Da n mass, as opposed to permethylaton or peracetylaton whch adds 14(n+m) or 42(n) Da (n s the number of free hydroxyl groups, m s the number of NH-Ac groups) to the mass of an olgosaccharde. In the case of larger olgosacchardes, our method s of partcular advantage, snce a large ncrease n mass upon dervatzaton would move the molecular mass from the readly accessble mass range where nstrument senstvty s n the subnanomole range. ABEE dervatzaton also produces a homogeneous product, n contrast to the mxtures often obtaned from use of permethylaton or peracetylaton procedures (30). When the ABEE dervatve s prepared, molecular weght and fragmentaton data can readly be obtaned by usng 1,g of olgosaccharde, even usng conventonal postacceleraton and a sngle-slt on multpler system. However, ths work has establshed that when computer-controlled mass wndow steppng s used wth multchannel array detecton, the level of sample requred can be reduced by as much as 100-fold usng a 4% mass wndow array (1000 channels). Desgn of on optcal system that wll permt full focal plane multchannel array detecton (31) wll ncrease the senstvty heren reported by yet at least a further order of magntude. Such Proc. Natl. Acad. Sc. USA 86 (1989) 6649 ultrahgh mass spectral senstvtes wll revolutonze future nvestgatons of the structural bology of membrane-bound glycoprotens such as growth factor receptors, whch cannot be readly obtaned n nanomole quanttes. It wll then be possble to sequence and structurally characterze protens, membrane protens, and ther posttranslatonal modfcatons from femtomolar quanttes. We thank Dr. A. L. Tarentno for generous provson of PNGase F. 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