Structural characterization of intact, branched oligosaccharides by high performance liquid chromatography and liquid secondary ion mass spectrometry

AUAL.YTICAL

BIOCHEMISTRY

169,

337-339

(1988)

Structural Characterization of Intact, Branched Oligosaccharides by High Performance Liquid Chromatography and Liquid Secondary Ion Mass Spectrometry’ KEJIANG,*.' BETH L.GILLECE-CASTho,* ANTHONY L. THOMAS H. PLUMMER.~] JAMES C. B>.RD,(/.’ SUSAN J. FISHER,*?$

JAMES W. WEBB,*,’

AND

TARENTINO./~

A. L. BURI.IUGAME*$.’

Received

August

3 I. 1987

We report results of a mass-spectrometric-based stratcg? for determining the detailed struttural features of N-linked oligosaccharides from glycoproteins. The method was used to characterize a series of intact. high mannose oligosaccharides isolated from human immunoglohulin M (IgM). The IgM was purified from a patient with Waldenstrom’s macroglohulinemia. The strategy included releasing the oligosaccharides hq digestion of the purified glycoprotcin \sith endoglycosidase H, separating the released oligosdccharides by high resolution gel filtration. and derivatizing the resulting reducing termini with the uv-absorbing moiety. ethyl p-aminobenzoatc. This particular derivative facilitates HPLC detection and provides centers for protonation and deprotonation enhancing liquid secondary ion mass spectra. Positive and negative ,011 spectra contained molecular species of similar abundance. However, fragment ion peaks yielding sequence information were significantly more prominent m the negative ion mass spectra. Furthermore. it was obvious that the fragmentation patterns differed substantially for linear and branched oligomers. For linear oligosacchandes. a smooth envelope of fragment ions was observed; from low to high mass there was an ordered decrease m ion abundance from both the reducing and nonreducing tcrmini. This pattern of fragment ions was not observed for branched oligosaccharides since in these cases fragments at certain masses could not arise b> single bond cleavages. Therefore, these fragments were either sigiticantly reduced in ahundancc or absent as compared with identical fragments formed from linear molecules. Importantly. 200 pmol of an oligosaccharide could be derivatizcd. separated. and detected by mass spectromctr). allowing identification of previously unreported minor components of the IgM oligosaccharides. Therefore. this experimental strategy is particularly useful for the purification and detalled structural characterization of low abundance oligtxxcharides isolated from hcterogeneous biological samples. c I’)XX .Acadrmlc Prr5s. Illi I(tY WORDS: structure: branched oligosaccharides: high performance lquid chrt,matogr:lph!: mass spectrometq. liquid secondary inn: Waldenstrom’s m~icroglohullncmi~~,

’ This work was suppwrted by Grant RR 0 16 I4 from the National Institutes of Health to the Bio-organic. Biomedical Mass Spectrometry Resource and Grants AM 17643 to the Liver Center Mass Spectrometry Core Facility (A. I.. Burlingame. Director). DE07244 (S. Fisher). and GM 3047 I (A. L. Tarentino).

’ Present address: Research Center for Eco-Environmental Sciences. Academia Sinica. P.O. Bo\ 934. Bciling. China. ’ Present addrccs: Veterans Administration Medrcal Center. San Frclncisco. C4 9-I I? I. ’ TI> \I holn correspondence should ix addrcsscd

338

WEBB

The structural characterization of mixtures of complex oligosaccharides such as those isolated from glycoproteins represents a formidable challenge to analytical biochemistry. NMR techniques are applied to these characterizations when sufficient quantities of oligosaccharide are available (1). If there is insufficient sample to perform NMR, alternative methods such as mass spectrometry are required. Other experimental strategies for analyzing mixtures of low abundance oligosaccharides often include radiolabeling the reducing termini with sodium borotritide. However, only limited information (e.g., relative retention times, exoglycosidase sensitivity) can be obtained. Thus, the development of advanced analytical methodologies is required. Eventually, methods for the analysis of heterogeneous samples from biological sources should permit the separation of all oligomers, including isomeric forms. In addition. these methods should also facilitate determination of the molecular weight, sugar sequence. and branching of each component. as well as linkage and anomerity. To this end, considerable attention has been focused on recent developments of ion or atom liquid matrix sputtering ionization mass spectrometry to gain information regarding the primary structure of intact oligosaccharides (3,3). Strategies for oligosaccharide structural characterization may also include derivatization of the reducing termini with suitable chromophores to permit HPLC detection. This is important since detection of small quantities of oligosaccharides precludes the use of the refractive index detector due to its relative insensitivity, as well as its incompatibility with gradient elution. Furthermore, derivatization often increases the kind of structural information which can be obtained by mass spectrometry. Improved sensitivity and enhanced fragmentation are especially useful when the available chromatographic methods do not readily achieve complete separation of all the major and minor components. Strategies employing dcriv,atization.

El- AL

HPLC separation. and mass spectrometry have been reported by other investigators using model oligosaccharides. For example. various oligosaccharides (n < 9) have been derivatized with I-amino pyridine (4,5) or 7-amino- 1-naphthol (6) and analyzed by positive ion FAB.’ CI, and tandem mass spectrometry. both with and without prior separation by HPLC. Sweeley and coworkers derivatized oligosaccharides with various uv-absorbing compounds including aniline and ethyl I?-aminobenzoate. The products fn < 12) were separated by HPLC and positive ion FAB mass spectra were rcported for several oligosaccharides (17 < 6). The authors concluded that this method could be applied to larger, more complex oligosaccharides (7). In this laboratory we are using a comprehensive strategy, incorporating recent advances in mass spectrometry, to separate and characterize protein-linked oligosaccharides. Important features ofthis experimental strategy include (i) enzymatic release of N-linked oligosaccharides using Endo H or PNGase F: (ii) derivatization of the oligosaccharides with ethyl p-aminobenzoate: (iii) separation of the products by HPLC; and (iv) structural characterization by cesium ion liquid matrix secondary ion mass spectrometry. We report use of this strategy to characterize intact oligosaccharides isolated from human IgM. as well as to obtain the molecular weights for previously unreported minor components. In the course of these experiments WC observed predictable differences in fragment ion abundances between linear and branched oligomers, suggesting that in addition to sequencing, branching information may be determined by the methods described in this paper.

’ Abbreviations used: FAB, fast atom bombardment: Cl. chemical ionization: Endo H. endoglycosidase H; PNGase F. peptidc-.~J-(~~~-acctyl-iJ-glucosamtnyl) asparagine amidase F: IgM, immunoglohulin M: ABEE. ethyl p-aminobcnzoate: ISIMS. liquid matrix secondary ion maw spectrometr) : DAB. 1,4-diaminobutane: GlcNAc. ,l’-acct~lglucclsamine.

CHARACTERIZATION

MATERIALS

AND

OF

BRANCHED

METHODS

Maltooligosaccharides (up to n = 10). ethyl p-aminobenzoate (ABEE). and sodium cyanoborohydride were obtained from Sigma Chemical Co. (St. Louis, MO). Glycerol and monothioglycerol were from Aldrich (Milwaukee, WI). Ultramark 162 1 and 443 were obtained from PCR Research Chemicals (Gainsville, FL). IgM was purified (8) from the plasma of a patient with Waldenstrom’s macroglobulinemia. Briefly, the glycoprotein was purified by precipitation at low ionic strength followed by solution in 0.15 M NaCl. Insoluble material was removed by centrifugation. This process was repeated four times, after which the solubilized IgM was further purified on Sephacryl S-200. High mannose and hybrid oligosaccharides were released by digestion of the glycoprotein with Endo H. Five fractions were collected from a Bio-Gel P-6 (-400 mesh) column (9) and designated a-e. Prqwrution ctnd separution c?f’ A BEE de riwtiws. The oligosaccharides were deriva-

tized with ABEE (7). Briefly. the sugar was dissolved in 10 ~1 of Hz0 and 40 ~1 of the reagent solution. Routinely, this solution was prepared by dissolving 165 mg of ABEE and 35 mg of sodium cyanoborohydride in 41 ~1 of glacial acetic acid and 350 ~1 of warm methanol. The glass vials were capped and heated at 80°C for 30 min. After the solution cooled, equal volumes of H20 and chloroform (generally from 250 to 500 ~1 each) were added to the reaction mixture and the derivatives were extracted into the aqueous phase. HPLC analyses were performed at 254 nm using a Beckman 322 MP chromatograph equipped with a Model 153 uv detector. The derivatized oligosaccharides were separated using a carbohydrate column (Alltech Assoc. Inc.. Deerfield IL; 300 mm X 4.1 mm i.d.) 01 1O-pm particle size aminopropyl-bonded silica and fitted with a guard column (50 mm X 4. I mm i.d.) containing the same stationary. uhase as the analvtical column. The lin.

339

OIJGOSACCHARIDES

ear mobile phase gradient consisted of an aqueous acetonitrile mixture from 20 to 600/ H?O. Samples used to determine the sensitivity of the method were purified using a Vydac C 18 column. The linear mobile phase gradient consisted of an aqueous acetonitrile mixture from 100 to 40% HzO. Elution in both systems was accomplished over 40 min at a flow rate of 1.0 ml/min. ,Ilcrs.c spcctronwtr~. Spectra were recorded using a Kratos MSSO-S double focusing mass spectrometer. This instrument was equipped with a high field magnet. an LSIMS source ( IO), a postacceleration detector ( IO keV), and positive to negative switching. Cesium ions bombarded the sample in a liquid matrix ( 11) at approximately 9 keV, and the secondary ions were accelerated to 8 keV. The magnet was operated in the field control mode typically scanning at 300 s/decade at a resolution between 1500 and 2500 depending on the mass range of interest. Mass calibration was performed using a mixture of Ultramark 1631 and 443. Nominal molecular weights were used to report all masses (e.g.. the fractional mass increase of 0.4-0.6 AMU for the Man5GlcNAc-ABEE through the MansGlcNAc-ABEE molecular ion species was not included). Prior to mass spectrometry. all samples were purified by HPLC as described above. From 0.5 to 1 ~1 of aqueous or methanolic solutions of the sample ( I- 10 pg/pl) was applied to a copper or stainless steel probe tip. and then a matrix (0.5-I .O ~1) of either glycerol or glycerol mixed with monothioglycerol ( l/ 1. v/v) was added. RESULTS

AND

DISCUSSION

h~altooliKo.~ac~~l~lridrs. Separation of ABEE derivatives of a maltooligosaccharide series (n = 4 to 10) on an aminopropylbonded silica column or an in situ modified silica column resulted in nearly baseline resolution of the oligomers (data not shown). Using the silica column and 1.4-diaminobutane in the mobile phase (7) (M + DAB + H)’ adducts were observed in the oligosac-

340

WEBB

ET AL.

(M+N.s)+

FIG.

I. Positive

ion LSIMS

charide mass spectra. Such amine modifiers have been recommended to preserve the efficiency of amino-bonded columns (12). However, in order to eliminate the possibility of adduct ion formation in the mass spectra, no organic amines were used in any of the subsequent oligosaccharide separations. Under these conditions the aminopropyl column exhibited a significant decrease in resolution over time, especially for higher molecular weight oligomers. Positive ion LSIMS (Fig. 1) of ABEE derivatized maltoheptaose (molecular weight of 1301) showed two prominent molecular species, (M + H)+ and (M + Na)+, at m/z 1302 and 1324. Losses of anhydrohexose residues are observed as fragment ions of low relative abundance for both the protonated and sodiated species; when excess sodium was present the sodiated species predominated. These anhydrohexose losses occurred by cleavage with a hydrogen rearrangement at each of the glycosidic bonds. resulting in a net loss of 162 mass units (Fig. 2, Series A). Associated with the loss of each hexose unit were additional ions at 18 mass units below (Fig. 2, Series B) and 28 mass units above (Fig. 2, Series C) the sodiated fragment (13). While the positive ion mode yields molecular species of high abundance, the relative abundance of fragment ions is generally too low to be analytically useful. This characteristic has

of ABEE-maltoheptaose

been reported previously for positive ion spectra of the 2-aminopyridine (4) and ABEE (7) derivatives of neutral oligomers. However, an abundant suite of fragments can be induced in the positive ion mode by collisional activation in both the ‘-aminopyridine (4) and ABEE derivatives (7). The MS/MS spectrum (data not shown) of the maltoheptaose-ABEE was obtained using FAB in the positive ion mode, selecting the (M + H)+ ion, nz/i 1302. for collisional activation which showed prominent Series A fragments in MS2.6 An additional reducing end tag, trimethyl p-aminoaniline, has been suggested recently for the purpose of obtaining fragment ion series from both the reducing and nonreducing termini using positive ion FAB mass spectrometry ( 14). Mass spectral data subsequently obtained in this laboratory indicate that neither the positive nor negative ion mode yields fragmentation of abundance comparable to that observed in the negative ion spectra of the ABEE derivatives described in this work (unpublished results). Negative ion LSIMS of maltoheptaoseABEE (Fig. 3) showed a prominent molecular anion (M - H)- at 1300 Da and an intense fragmentation pattern corresponding b MS/MS (unpublished

data kindly results).

provided

by Dr.

K. Biemann

CHARACTERIZATION

OF

BRANCHED

341

OLIGOSACCHARIDES

or I

1 +H cl, -Ii

b 0

/”

R= OH or NHCOCt$

+

-

+

R

+H I+

Series B +

>

HO

7il+ CYOH

.I

/c + Ct$OH

Series C

C&OH

7’

Series D /

RO

I

OH

FIG. 2. LSIMS oligosaccharide radical mechanisms ( 15).

fragmentation

pathways.

to Series A. Thus, these spectra contained extensive sequence information as compared to spectra obtained in the positive ion mode without collisional activation. Occasionally, (M + Na - 2H)) and/or (M + Cl)) adduct anions were also observed, depending on the salt content of the sample. Previous work on the use of this derivative reported only the positive ion spectra which were dominated in many cases by the observation of relatively intense glycerol and sodiated glycerol matrix cluster ions (7). The 2-aminopyridine derivative gave little or no information in the negative ion mode (data not shown). Another, less intense, fragmentation series (Fig. 2, Series D) was observed with charge retention on the nonreducing terminus of ABEE-derivitized oligosaccharides. This fragmentation

CYOH

These mechanisms

may proceed

similarly

hy

was not observed in the positive ion spectra and appears to be due to a rearrangement in the reducing end moieties (15). The lowest mass ion fragment of this series (D) was observed at m/z 22 1. while the larger fragments appeared sequentially every 162 mass units. The origin of these fragments was confirmed by analyzing the p-amino-[ I’, 1’,2’,2’,2’‘HIethyl benzoate derivative of maltoheptaose using negative ion LSIMS (Fig. 4). The Series A ions were all shifted by five mass units, whereas the Series D ions did not change. Taken together these data (Figs. 3 and 4) indicated that fragments from both the reducing and nonreducing termini of this linear molecule gradually decreased in abundance from low to high mass. We have investigated the overall sensitiv-

342

WEBB

ET AL.

600

5 z

(M-HI. 0

(M*Na-ZH)

FIG. 3. Negative

ion LSIMS

of ABEE-maltoheptaose

333

FIG. 4. Negative

ion LSIMS

(M-H) 1305

ofthef-amino-[

I’. I ‘.2’.1’.2’-LH]ethyl

bcrvoate

derivative

of maltoheptaose

CHARACTERIZATION

OF

BRANCHED

ity obtainable employing the ABEE derivative in two ways. First, we determined the minimum amount of derivatized oligosaccharide which could be analyzed by LSIMS. ABEE-maltoheptaose was purified by HPLC and used to prepare standard solutions. While the molecular anion was detected from 4 pmol of ABEE-maltoheptaose. -40 pmol was necessary to observe an interpretable fragmentation pattern. Next. we determined the minimum amount of oligosaccharide required for all the steps in the procederivatization. HPLC dure, including separation. and cesium ion LSIMS: a molecular ion was obtained from 300 pmol maltoheptaose (Fig. 5). These results together with those of Wang ~‘2al. (7) and Gillece-Castro cjt al. (16) indicated there are several advantages to analyzing ABEE-derivatized oligosaccharides: (i) ease of preparation: (ii) stability: (iii) enhanced sensitivity in LSIMS as compared to underivatized carbohydrates: (iv) minimal increase in molecular weight; and (v) enhanced fragmentation in negative ion mode. Consequently, the IgM oligosaccharides were analyzed as ABEE derivatives. IgAl oliyosa~c.lzurides. Next, we analyzed the fraction of IgM oligosaccharides which

._ /

,~

I

FIG. 6. HPLC rides. A

343

OLICOSACCHARlDES

separation

of IgM-ABEE

oligosaccha-

1300

FIG. 5. Negative ion LSIMS of 100 pmol of ABEEmaltoheptaose. (A) The spectrum obtained from a sample of 200 pmol of maltoheptaose which was derivatized and isolated by HPLC. Only a third of the sample was applied to the LSIMS probe. (9) The spectrum obtained from a 400.pmol preparation of ABEE-maltoheptaose. One-half of the sample was applied to the LSIMS probe tip.

were released by digestion with Endo H. This subset of oligosaccharides had been separated into five fractions (a-e) by gel filtration chromatography. Each fraction was derivatized with ABEE and subjected to further separation by HPLC. The resulting chromatograms suggested that each of the five original fractions contained at least one major and several minor components (Fig. 6). This apparent heterogeneity was confirmed using positive ion LSIMS to obtain a molecular weight profile of each HPLC peak. The proposed composition of each peak from all fractions is given in Table 1. In general. individual HPLC peaks contained a major component and many minor compo-

344

WEBB

ET AL.

TABLE

I

MOLECULAR ION SPECIESFROM P~SWIVE ION LSIMS OF TW ABEE DERIVATIVE HIGH MANNOSE OLIGOSACCHARIDESFROM IgM a. I. 7 3: 4.

Nothing detected Man, GlcNAc2 (I 546); U (1709); II ( IZYY) Man, GlcNAc, ( 1870); Man, GlcNAc ( 1667): Man, GlcNAc,” ( 1827): U ( I3YX): II ( 1277) Man, GlcN.4~ ( 1820)

b. I. Mans GIcNAc, ( 1406): Man, GlcNAc2’ ( 1719): U ( I 137) 2. u ( 1709) 3. Man, GlcNAc ( 1667) C. 1. 1. 3. 4.

Man, Mani Man, Man,

GlcNAc,” ( 1382): Man, GlcNAc, ( 1546); Man, GlcNAc,” ( i 544): U ( 1074) GIcNAc? (I 384): L ( 1074) GlcN.4c (1505) GlcNAc ( 1667)

d. I. u (1385): u (1383) 2. Manh GlcNAc” ( I341 ): U ( 13x5); LJ ( 108X) 3. Manh GlcNAc ( 1343) e. I. U(1273) 2. Man, GlcNAc” (I 179) 3. Man, GlcNAc ( I I8 I ) ,V’oi~. LJ, Unknown origin. Sodium adduct ions were observed in vaning relative abundance. “ Schifl’s Base (unreduced ABEE derivative).

nents. Usually. minor components were due to incomplete chromatographic resolution of adjacent peaks or the presence of some unreduced Schiff bases. Retention times increased proportionately with the number of hexose residues. However, for oligomers containing the same total number of carbohydrate residues,any oligomer with more IVacetyl hexosamines eluted prior to the oligomer with the same number of neutral sugars(Fig. 6 and Table 1). With regard to the major IgM oligosaccharides, protonated molecular species were consistent with the structures previously published by Chapman and Kornfeld (17. 18). as well as with unpublished ‘H NMR dala.7 This NMR information was used to identify linkage positions and branch points

’ ‘H NMR data (unpublished). in collaboration with Drs. R. B. Trimble and P. H. Atkinson.

of major oligosaccharides (Table 3). Positive ion spectra of the minor HPLC fractions gave interesting evidence of other, previously unreported components. For example. fraction a-3 contained a Man*GlcNAc:-ABEE oligosaccharide. Since this oligosaccharide was releasedby Endo H, only one of the glucosamines originated from the chitobiosc core. In addition. fraction a-3 contained an unreduced Schiff base formed from MangGlcNAc, A Man8GlcNAcl-ABEE oligosaccharide. the major component of fraction b-3. was also evident. The origin ot peaks at m/: 1277 and 1398, one massunit higher than a Man,GLcNAz-ABEE oligosaccharide. has not been determined. The major components observed by HPLC (Fig. 6a-6e) were analyzed using negative ion LSIMS. These data are presented in Figs. 7-9 and summarized in Tables 2 and 3. Importantly. sequence information, from both the reducing (Series A) and nonreduc-

CHARACTERIZATION

-

w

,

”

OF

BRANCHED

OLIGOSACCHARIDES

345

ing (Series D) ends of the oligosaccharide. was apparent. For linear oligosaccharides (see Fig. 3 and 4) fragment ions from both the reducing and nonreducing termini grddually decreased in abundance from low to high mass. This ordered decrease in ion abundance was not observed for branched oligosaccharides (Figs. 7-9) since in this case fragments at certain masses could not arise by single Series A fragmentations. Therefore, the relative abundance of these ions was either significantly reduced or absent as compared to identical fragments formed from linear molecules. For example. the negative ion LSIMS analysis of HPLC sample b-3 (Fig. 8) showed an intense molecular anion at 1665. Series A ions for losses of one, two, three. or eight mannoses were evident (Table 2). as well as a less intense ion corresponding to the loss of four mannose residues (trr/: 10 17). Series D ions for fragments containing one, two. three, or four mannose residues (see Table 2 and Fig. 8) were also observed. These data suggested that the branched residues in the molecule suppressed the formation of fragments consisting of five. six, or seven mannose residues, evidence that the major component is formed from a triantennary core (see Table 3). Furthermore, the molecular mass together with the observed losses of one. two, three. or four mannose residues from the nonreducing termini suggested that two possible branched structures may be present, one with residues at positions 6, 7, 8. 9. and 11, and the other with a mannose at position 10 instead of 9 (see Table 3. Structures II and III). These two structures are consistent with the major and minor components observed by ‘H NMR. These data were typical of those obtained for all the fractions analyzed by LSIMS. The structures for the major and minor IgM oligosaccharides obtained using this method were consistent with those previously published by Chapman and Kornfeld (17.18) as well as with unpublished ‘H NMR data.’ However, some spectra suggested the presence of minor components which were not

346

WEBR

I AH1.E

Man IQ)

3

-~-

2 Man (6) \

\ ; Man

; Man (41

/ Man VI

Man --2 1111

Man--nMan 181

-

AL

Man

Man --2 (101

Man

El’

Man

\ ; Man-

aGlcNAc-

--2

Man/

\ ; Man -

ABEE

/,

ABEE

4GlcNAc-

ABEE

aGlcNAc-

ABEE

e,GlcNAc-

ABEE

VII

/

Man--2Man

aGlcNAc-

(5)

2 Man \ ; Man Man/

\ ; Man-

Man

--2

/

Man-nMan

dGlcNAc-

; Man-

ABEE GlcNAc-2Man

II

Man

Man \

‘63 Man Man

Man /

--2

/

VIII

\ ; Man

Man/

\ 6 Man -

aGlcNAc-

\ ,” Man-

ABEE /

2 Man

-zMan--2Man

Man

--2

/

Man-nMan

III

-

\

2 Man /

Man

\ 6 Man -4GlcNAc-

--2

M.,/‘Ma”\ ; Man -

ABEE

P Man --2

Man

--2

Man /

Ma”

IV

Man j3

X

\ ; Man Ma” /

Man\ ; Man -

Man --2

4GlcNAc-

16

3 Man

Ma” /

\ ; Man -

4GlcNAc-

ABEE

/ 2 Man -2

Man’

; Man Man /

V

Man /

\

ABEE

Ma”

IVO[C~ Numbering

/

Mall

Man 16

Man -

IX

Ma”

Man,

‘63 Man Man

/

VI

used for structure

I applies

to all structures.

XI

4GicNAc-

ABEE

CHARACTER!ZATION

OF

BRANCHED

OLIGOSACCHARIDES

347

FIG. 7. Negative ion LSIMS of fraction a-4 (see Fig. 6) ofthe IgM-ABEE oligosaccharidcs. The spectrum showed an intense molecular amon at 1827. Series A ions for losses of one. two, three, or tivc mannoses were evident (Table 2). as well as a less intense ion corresponding to the loss of four mannose residues (FH/; I 179). Series D ions for fragments containing one. two. three. or five mannose residues were also observed. These data suggested that the branched residues in the molecule suppressed the formation of fragments consisting of six or seven mannose residues, evidence that the major component is formed from a triantennary core (see Table 3. Structure I).

detected by NMR. For example, the intensity of the 855 ion, corresponding to the loss of five mannose residues. present in the spectrum of fraction b-3, suggested the presence of a minor component not detected using NMR. A triantennary structure (Table 3, Structure IV) with mannose residues substituted at positions 6, 7, 8, 9 and 10, was consistent with this data and was previously

FIG. 8. Negative

ion LSIMS

of fraction

reported by Chapman and Kornfeld ( 18). In addition, other ions of low relative abundance were also evident. The origin of these ions is not known at the present time. However. one of two possibilities seems most likely. First, assuming that the internal energy imparted to the molecular anion is sufficient to induce only one elimination per molecule, no corresponding ion current

h-3 (see Fig. 6) of the IgM oligosaccharides

WEBB

ET AL

Rc;. 9. Negative ion LSIMS of fractions c- 2, c-3, d-3. and e-3 (see Fig. 6) of the IgM oligosaccharides. (A) The spectrum obtained from fraction c-2. An intense molecular anion at I382 and prominent fragments listed in Table 2 were consistent with Man5GlcNAcz-ABEE (Table 3, Structure VIII). (B) The spectrum obtained from fraction c-3. An intense molecular anion at I503 and the prominent fragments listed in Table 2 indicated that the major component was Man,GlcNAc,-ABEE (Table 3. Structure VI). (C) The spectrum obtained from fraction d-3. An intense molecular anion at I341 and the prominent fragments listed in Table 2 indicated that the major component was Man,GlcNAc,-ABEE (Table 3. Structure IX). In addition, the ion m/z 693 indicated that Structure X (Table 3) could be a minor component of this fraction. (D) The spectrum obtained from fraction e-3. An intense molecular anion at I 179 and prominent fragments listed in Table 2 were consistent with Man,GlcNAc,-ABEE (Table 3. Structure XI).

should be detected where branching is present. In this case, minor peaks would in fact reflect the presence of minor branched isomers. Second, as suggested by Dell and Ballou (19), these ions may arise from double cleavages. Further work is in progress aimed at a definitive resolution of this point. In summary, preparation and analysis of ABEE-oligosaccharides using the experimental strategy described represents an important methodological advance in the structural analysis of carbohydrates. Using as little as 200 pmol of oligosaccharide we were able to readily obtain information usually requiring 10 times this amount of sample (2,3).

Formation of the ABEE derivative for oligosaccharides possessing a free reducing terminus confers several important characteristics of analytical advantage for studies of structures and heterogeneity of N-linked glycosylation on glycoproteins. These include (i) high sensitivity in derivative preparation, 200pmol sample size; (ii) a chromophore for HPLC separations: (iii) observation of primarily MH+ in the positive ion mode for determining a molecular weight profile; and (iv) production of a fragmentation pattern in the negative ion mode which provides sequence and branching information. These results indicated that the method described

CHARACTERIZATION

OF

BRANCHED

can be effectively auDlied to determine the structure of low abundance oligosaccharides isolated from heterogeneous biological samples.

I. Sweeley. C. C.. and Nunez. H. A. (1985) .-1w1w Bmhern. 54, 765-80 1. 2. Burlingame. A. L.. Baillie. T. A.. and Derrick. ( 1986) heal. C‘hern. 58, I hSR-2 1 I R. 3. Egge. H.. and Peter-Katalinic. J. ( 1987) ,‘lla.cr from Rev. 6, 331-393. 4. Carr. S. A.. Reinhold. V. N.. Green. B. N.. and J. R. (1985) Bi~~~,?c~d. ,2/cts.c .Spcc~rc~w.

9. Byrd. J. C.. Tarentino, A. L., Maley. F., Atkinson. P. H.. and Trimble, R. B. (1982) J. Biol Chern. 257, 14657-14666. 10. Falick. A. M., Wang. G. H.. and Walls, F. C. ( 1986) .-lrd C‘hern. 58, 1308- I3 1 I. I I. Aberth. W.. Straub. K. M.. and Burlingame. A. L. (1981)

REFERENCES Rm. P. J. Spw Hass. 12,

288-295. 5. Reinhold.

V. N. (1986) irl Mass Spectrometry in Biomedical Research (Gaskell. S. J.. Ed.). Chapt. 1 I. pp. 181-213. Wiley. New York. 6. Cole% E.. Reinhold. V. N.. and Carr. S. A. ( 1985) Carbolzwh Rc.s. 139, l-l I. 7. Wang, W. T.. LeDanne. N. C.. Jr., Ackerman, B., and Sweeley. C. C. (1984) .4wI. Br~~~hw~. 141,

~‘lwn

A. L., Plummer. 7. H.. Jr.. and Male);. Bio&wi.str~~ 14, 55 16-5513.

F.

.-id

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C. E. (1983)

Cwlwhrdr.

Hrs.