JOURNAL OF MASS SPECTROMETRY, VOL. 31, 1109-1118 (1996)

Does Matrix-assisted Laser Desorption/Ionization Mass Spectrometry Allow Analysis of Carbohydrate Heterogeneity in Glycoproteins ? A Study of Natural Human Interferon-y

Ejvind Mart2 Department of Molecular Biology, Odense University, Campusvej 55,5230 Odense M, Denmark

Timo Sareneva and Ilkka Julkunen Department of Virology, National Public Health Jnstitute, Mannerheimintie 166, FIN-00300 Helsinki, Finland

Peter RoepstorffT Department of Molecular Biology, Odense University, Campusvej 55,5230 Odense M, Denmark

Interferon-y (IFN-7) is a dimeric, secretory glycoprotein produced by T-lymphocytes. The glycan micro- heterogeneity of natural human IFN-y was characterized by matrix-assisted laser desorption/ionization mass spec- trometry (MALDI/MS) combined with glycosidase digestion. The glycan structures at the two potential glycosylation sites, asparagine 25 and 97, differ in composition and heterogeneity. The glycan at Asn 25 consists of a mixture of hybrid structures and fucosylated complex bi-, tri- and tetra-antennary structures, whereas the glycan at Asn a7 is more heterogeneous and consists of a mixture of high mannose structures, hybrid structures and unfucosylated complex bi- and tri-antennary structures. The contribution to the observed glycan heterogeneity by prompt and metastable fragmentation was evaluated by treatments with different exoglycosidases and by compari- son of linear, reflected and delayed extraction MALDI/TOF mass spectra. Heterogeneity observed with the matrices a-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid and 2,4,4-trihydroxyacetophenone was com- pared. Most of the heterogeneity can be attributed to native structure diversity and only to a minor extent to mass spectrometric fragmentation such as fragmentational loss of sialic acid residues.

KEYWORDS: matrix-assisted laser desorption/ionization mass spectrometry; interferon-y; post-translational modification ; protein glycosylation; carbohydrate heterogeneity

LNTRODUCTION

The carbohydrates linked to specific asparagine residues in eukaryotic proteins are often a mixture of closely related oligosaccharides, often referred to as glycosyla- tion microheterogeneity. The pattern of oligosaccharide structures presented at a glycosylation site is not random, but is characteristic for the glycosylation site, the protein, the cell type and the physiological condi- tions in the cell (reviewed in Refs 1-3). The structures of oligosaccharides in a glycoprotein can have profound effects on its biological properties, such as protein folding, oligomerization, protein targeting, overall turn- over rate and susceptibility to proteases and regulation of protein-protein interactions (reviewed in Ref. 4). If each individual glycostructure adds distinctive features to the protein then the controlled variation of presented glycostructures may in a population of glycoprotein molecules lead to a controlled functional diversity of the protein. In many proteins, however, the carbohydrate heterogeneity and the roles of the carbohydrates are diverse, complex and not very well understood. This is

t Author to whom correspondence should be addressed.

partly caused by lack of information because many gly- coproteins are available only in low quantities and because traditional methods for structural analysis of carbohydrates, such as NMR and fast atom bombard- ment (FAB) mass spectrometry (MS), require large amounts of the glycosylated protein and mainly show the major components in a mixture of carbohydrates. At present, more sensitive methods for analysis of car- bohydrates are being developed, including high- performance anion-exchange chromatography with pulsed amperometric detection,' capillary zone electro- phoresis,6 matrix-assisted laser desorption ionization (MALDI) MS,7-9 liquid chromatography/electrospray ionization MS'"' ' and enzymatic methods such as the reagent array analysis method.I2

Interferon-y (IFN-y) is an important immuno- modulatoric glycoprotein and is of considerable interest owing to its potential therapeutic use. IFN-y is produc- ed by T-lymphocytes and natural killer cells after induc- tion with mitogens or specific antigens. It has also been produced recombinantly in a number of expression systems, i.e. Chinese hamster ovary cells, Escherichia coli, insect cells and transgenic mice.' 3-'5 The mature IFN-y molecule contains 143 amino acids and two N - glycosylation sites with the consensus sequence Asn--X- Thr/Ser, with Asn being asparagines 25 and 97,

CCC 1076-5174/96/101109- 10 0 1996 by John Wiley & Sons, Ltd.

Received 16 April 1996 Accepted 26 June 1996

1110 E. MBRTZ ET AL.

Table 1. Glycosidases used

Glycosidase Origin Supplier’

a- Fucosidase a- Mannosidase B- Galactosidase Endoglycosidase F Endoglycosidase H N-Acetyl-~-D-glucosaminidase Neuraminidase N-Glycosidase F

Bovine epididymis Jack bean Diplococcus pneumonia Flavobacterium menigosepticum Streptomyces lividans Diplococcus pneumonia Arthrobacter ureafaciens Flavobacterium menigosepticum

OG OG B M BM BM BM BM B M

BM = Boehringer Mannheim (Germany); OG =Oxford Glycosystems (UK).

Specificity

al-6 > al-2, ul-3, al-4 al-2, al-3, al-6 B1-4 Complex, hybrid, mannose Hybrid, mannose B1-2 a2-6> a2-3, a3-6 Complex, hybrid, mannose

Buffer

25 mM NH,Ac, pH 6.0 25 mM NH,Ac, pH 6.0 25 rnM N H d c , pH 5.0 25 mM NH4Ac, pH 6.0 25 mM NH4Ac, pH 6.0 25 mM NH,Ac. pH 5.0 25 mM NH4Ac, pH 5.0 25 mM NH,HCO,, pH 7.8

respectively. The natural protein is partly glycosylated as seen by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), which shows three bands at molecular masses 16, 20 and 24 kDa, presum- ably corresponding to the non-glycosylated, mono- glycosylated and diglycosylated protein, re~pectively.~~~” Most of the dimeric IFN-y molecules (-95%) are glycosylated at least at two of the four potential glycosylation sites.” The glycans on natural IFN-y have been demonstrated to consist of complex sialylated biantennary structures with some heter- ogeneity in the content of sialic acid and fucose.lg Pre- liminary investigation of the site-specific glycan structures on the natural protein was investigated pre- viously after separation of the different glycosylation forms by SDS-PAGE.” These investigations gave infor- mation about the major glycan structure on Asn 25, but only limited information about the structures on Asn 97. The glycans of recombinant IFN-y have been char- acterized and their complexity seems to correspond to the glycosylation pathways available in the given expression system.20 However, if the recombinant pro- teins are to be used therapeutically, it remains to be established how closely their glycan structures resem- bles those of the natural protein. In the present study we used MALDI/MS in combination with specific gly- cosidases to elucidate and characterize the glycan struc- tures and heterogeneity in natural human IFN-y.

NaCl buffer (pH 7.3) and eluted from the matrix with 2 M NaCl in 150 mM ammonia solution (pH 10.7), fol- lowed by neutralization with 1 M phosphate buffer (pH 6.0). The resulting protein preparation contains a mixture of non-, mono- and diglycosylated IFN-y. The protein content of the highly purified IFN-y preparation was 0.4 mg ml-’ and the specific antiviral activity was 2 x 10’ IU mg-l protein.

Preparation of glycopeptides

Native IFN-y (8 pg) was desalted by high-performance liquid chromatography (HPLC) on an Applied Biosys- tem 130A reversed-phase HPLC system equipped with a 2 mm id. C, Brownlee column using a buffer system of (A) 0.1% trifluoroacetic acid and (B) 0.08% TFA in 90% acetonitrile. The purified IFN-y was dissolved in 100 pl of digestion buffer (50 mM NH,HCO, (pH 7.8)- 0.5 M urea) and digested with 0.1 pg of modified trypsin (Promga) for 3 h at 37 “C. The generated peptides were separated by HPLC using a 2 mm i.d. C,, Brownlee column. The collected HPLC fractions were analysed by MALDI/MS and the masses correlated to the IFN-)I sequence. Fractions containing glycosylated peptides were identified from their complex mass spectra caused by heterogeneity of the glycan, i.e. mass differences of 162 and 291 Da corresponding to hexose and sialic acid residues, respectively.

EXPERIMENTAL Mass spectrometry

Purification of interferon-y

Natural IFN-.)I was produced by human leukocyte cul- tures and partially purified by ion exchange (CM-Sep- hadex) followed by further purification in an immunoafinity matrix with monoclonal anti-IFN-y antibodies.* 1.22 Briefly, lectin-induced leukocyte cell culture supernatants were dialysed against 30 mM phos- phate buffer (pH 6.7) and the proteins were allowed to bind to CM-Sephadex C-50 (Pharmacia). After washing with the binding buffer, the bound material was eluted with 20 mM phosphate buffer (pH 8.0) containing 1 M NaCl. Further purification of the protein was achieved by using monoclonal anti-IFN-y antibodies coupled to Eupergit C immunoadsorbent (Rohm Fharma). The bound IFN-y was washed in 20 m~ phosphate-0.5 M

MALDI/MS was performed on a Bruker reflex time- of-flight instrument using the software LaserOne (programmed by M. Mann and P. Mortensen, EMBL, Heidelberg, Germany) for data acquisition and pro- cessing. Thin-film matrix surfaces were prepared using the fast evaporation technique2, from ol-cyano-4- hydroxycinnamic acid (4HCCA) (Sigma) dissolved in acetone-water (99: 1) (30 pg pl-’) or from a saturated solution of 2,4,6-trihydroxyacetophenone (THAP)24 (Aldrich) dissolved in methanol. A 0.5 p1 volume of the analyte (0.1-1 pmol pl-’) was deposited on the matrix surface with 0.5 pl of 2% TFA and dried before washing repeatedly with water to remove salts from the matrix crystals. Spectra were obtained by averaging 100 -200 single-shot spectra and calibrated using external cali- bration or matrix peaks as internal c a l i b r a n t ~ . ~ ~

HETEROGENEITY OF INTERFERON-7 GLYCANS 1111

N-acetylneuraminic acid (291 26)-

N-acetylglucosamine (203.20)-

Galactose (1 62.14)

N-acetylglucosamine (203.20)-

Mannose ( 1 62.1 4)

Fucose (1 46.1 4)

-Am-X-Ser/Thr-

Figure 1. Example of a hybrid carbohydrate structure. Monosac- charide residues are given on the left with average molecular masses in parentheses. Glycosidase cleavage sites are indicated with arrows.

Glycosidase digestion

Sequential glycosidase digestion was performed using the glycosidases described in Table 1. Digestion was carried out in 10-20 pl of buffer at 37 "C and aliquots of 0.5 1.11 were removed after 24-48 h of digestion and analysed by MALDI/MS. Sequentially other glycosi- dases were added and aliquots analysed by MALDIJ MS. The mass shifts corresponding to removal of monosaccharide units were monitored to elucidate the oligosaccharide sequences, as illustrated in Fig. 1.

RESlL JLTS

Alternatively, MALDI/MS was performed on a Per- Septive Voyager Elite instrument equipped with a delayed extraction ion sourcez6 and a camera for observation of the matrix crystals. Samples were pre- pared with the traditional dried droplet matrix prep- aration method using the 2,5-dihydroxybenzoic acid (DHB) matrix (Hewlett-Packard, Palo Alto, CA, USA) by mixing 0.5 pl of matrix solution with 0.5 pl of sample dissolved in 30% acetonitrile-2% TFA on the target. Ions were accelerated at an acceleration voltage of 20 kV after a delay time of 250 ns.

Identification of glycosylated peptides

IFN-y was digested with trypsin and the resulting pep- tides were separated by HPLC. Analysis of the HPLC fractions by MALDIJMS showed the presence of gly- cosylated peptides in four of the fractions. The MALDI mass spectra obtained for two of the HPLC fractions are shown in Fig. 2. Based on the mass spectra, these peptides were identified as glycosylated peptides, because heterogeneity of the carbohydrates was seen by mass differences of 162 and 291 Da corresponding to

Mass [Da] Mass [Da] Figure 2. Linear MALDI mass spectra of the two HPLC fractions containing the peptides glycosylated at (A) Asn 97 and (6) Asn 25. The peptides were deglycosylated with N-glycosidase F (C and D, respectively) and the peptides identified. The spectra were obtained using 4HCCA as matrix. The masses assigned in the spectra are MH' values.

1112 E. MORT2 ET AL.

hexose and neuraminic acid residues, respectively. The peptides were treated with N-glycosidase F, which cleaves off Asn-linked carbohydrates and converts the Asn to Asp. The peptide masses were determined by MALDIiMS [Fig. 2(c) arid (d)] and correlated to the protein sequence. In the spectrum in Fig. 2(c) a new peak appeared at m/z 2159.3 corresponding to residues 90-107 (calculated m/z for MH' 2159.3) containing the Asn 97 gl ycosylation site. In addition to the glycosylat- ed peptide, the MALI31 mass spectrum of this HPLC fraction showed three unglycosylated peptides at miz 2286.8 (residues 90--108), 23 14.8 (residues 89-107) and 2442.7 (residues 88-107). In the spectrum in Fig. 2(D) a peak appeared at mlz 2255.8 corresponding to residues 14-34 (calculated m/z for MH' 2254.5) containing the glycosylation site Asn 25. The two glycosylated peptides eluting in other HPLC fractions also contained Asn 97 (residues 89-107 and 88--107) and were caused by incomplete tryptic cleavages in the sequence "KKKR9'.

Elucidation of glycan structures

The IFN-.)I glycan structures were elucidated by sequen- tial digestion with specific exoglycosidases and by moni- toring the resulting mass losses by MALDI/MS. Complex and hybrid carbohydrates were digested with two series of exoglycosidases: (i) neuraminidase fol-

lowed by fi-galactosidase and (ii) j3-galactosidase fol- lowed by N-acetylglucosaminidase. High mannose structures were digested with a-mannosidase.

Asparagine 97. Selected spectra from elucidation of the glycan structure at Asn 97 are shown in Fig. 3. The molecular masses of the glycans were calculated by sub- tracting the molecular mass of the deglycosylated peptide from the masses observed for the native gly- cosylated peptide [Fig. 3(a)]. Based on the knowledge about other mammalian N-linked glycans and the determined glycan masses, the structures of 18 different carbohydrates could be suggested from the spectrum in Fig. 3fa) (shown in Fig. 4, centre row). To verify the presence of these structures, the glycopeptide was treated with glycosidases and the subsequent mass losses were determined by MALDI/MS. The presence of seven structures containing sialic acid residues was veri- fied by digestion with neuraminidase and by observing mass losses of 291 Da or multiples thereof correspond- ing to removal of sialic acid residues [compare Fig. 3(a) and (b)]. The following mass losses were observed: m/z 4364.914072.7 to 3782.5; 4222.1 to 3927.4; 3703.2 to 3417.8; 4438.3 to 4147.9; 3868.4 to 3579.2 and 4032.1 to 3741.2 (illustrated in Fig. 4, digestion series A). The presence of three of the 18 structures could not be veri- fied after the neuraminidase digestion (indicated 'Not Obs.' in Fig. 4). Structures with terminal galactose resi- dues were further digested with 8-galactosidase

A '2 Native structures 16

n 1 II

Neurarninidase B

Mass [Da]

P-galactosidase + N-acetylglucosaminidase

D ? a-mannosidase 2

Mass [Da] Figure 3. Linear MALDI mass spectra of the peptide glycosylated at Asn 97 treated with glycosidases. The native carbohydrate structures (spectrum A) were digested sequentially with neuraminidase (spectrum B) followed by p-galactosidase. Alternatively, the native structures were digested with p-galactosidase followed by N-acetylglucosaminidase (spectrum C). To verify the presence of high mannose structures, the native structures were digested with a-mannosidase (spectrum D). The spectra were obtained using THAP as matrix. The oligosac- charide structures that are deduced from the digestion series are illustrated in Fig. 4.

HETEROGENEITY OF INTERFERON-1 GLYCANS 1113

Putatwe ~ l v c g l - structures o n A n s 9 7 mestio n series A Diqestion series €3

P-galactosidase t Neuraminidase c Nativestructures4 fl-galactosidase - N-acetylglucosaminide

Not oh.

1- 3782.5 (3781.8)

< 3 -< 391 2.8 (391 0.D) 4072.7 (4073.1)

4

-< 3708.3 (3707.8)

< - 3457.5 (3457.5)

Not obs.

Not obs.

(4) / 4222.1 (421 9.1 )

3927.4 (38273) 0 3927.9 (39278)

Not obs.

1- 3417.8 (3416.5)

3703.2 (3707.8)

3414.4 (3416.5) - I+ _Hcgl 3253.4 (3254.4) -1 NotOb.

< 3256.5 (3255.7)

- 4 3051.3 (3051 .l)

Not Oh. I --z? 4438.3 (4438.4)

Not obb.

-+E 4147.9 (4147.2) -=-2

3bBo.8 (3660.7)

4148.1 (4147.2) - I_ c --z 3661.1 (3660.7) +-+TI- C

3820.8 (3822.9) 3821.5 (3822.01 J

< 3869.8 (3869.9)

1 -

3868.4 (3869.9) --c 3415.8 (3416.5)

-< 3579.2 (3578.6)

3369.6 (386S.9)

3213A (3213.3) 4 2418.1 (3416.5) 3577.8 (3578.6)

-F+ 4032.1 (4032.0)

- - 3739.4 (3740.8)

Noi obs. Not Ob6 - 3741.2 (3740.8)

Not Obs. -* 3375.5 (3375.4) 3580.4 (3578.6)

Not obb.

Not obb.

c a-mannosidase

3213.1 (3213.3) 3049.9 (3051.1 )

3535.2 (3637.6)

3376.4 (3375.4)

Not obs.

Figure 4. Schematic illustration of the oiigosaccharide structures at Asn 97. The structures are deduced from the molecular masses deter- mined for the native glycan (centre row) and the digestion with neuraminidase followed by p-galactosidase (digestion series A), and B-galactosidase followed by N-acetylglucosaminidase (digestion series B). Structures deduced from digestion with or-mannosidase are shown in the inset. The determined and caiculated m/z values are given below the structures (calculated m/z values in parentheses). The symbols for the monosaccharides are shown in Fig. 1.

1114 E. M0RTZ ET AL.

Putative plvcan structures o n Asn 25

Dieestio n series A Dbestion series B B-galactosidase f- Neuraminidase - Native structures -+ B-galactosidase 4 N-acetylglucosaminidase

3899.1 (38ee.8) -1 -< 4152.7 (4152.2) 431 6.8 (431 4.3) 3950.0 ( W . 0 )

4023.q4023.1)

Not obs.

4023.5 (4022.1)

- T-c kE.8) Not obs. \ 4 Not obs.

3854.2 (3949.0)

s l r

3485.5 (3495.6)

3 - r +-I -3-f 3292.8 (3292.4) No1 OL.. 3496.6 (3495.6)

i 3497.0 (3495.6) J

NOlm6.

Not obs.

Not Obs.

Not obs.

Not obs.

Not obs. r < 4008.2 (4007.b) 4168.8 (4189.1)

4169.9 (4169.1) 4007.5 (4007.0)

4971.7(4970.9)

4679.6 (4870.6) 4356.3 (4355.4)

3902.3 (3902.0)

4753.6 (4753.7) 5043.9 (5045.1) 4108.5 (4105.2)

Not Obs. * c- < Nolobs.

3982.4 ( g 2 . 0 ) 4271.8 (4273.2)

Figure 5. Elucidation of the oligosaccharide structures at Asn 25. Performed as described for Asn 97 (see also Figs 3 and 4).

(illustrated in Fig. 4, digestion series A). Mass losses of 162 Da corresponding to the removal of galactose resi- dues was observed for the expected structures, except for a hybrid structure (m/z 3740.8). One fucosylated structure (m/z 3729.9) and two high mannose structures expected at mlz 3375.4 and 3537.6 were not observed after the 8-galactosidase digestion.

To confirm further the presence of the 18 structures, the Asn 97-containing peptide was treated with @- galactosidase followed by N-acetylglucosaminidase. On @-galactosidase digestion, mass losses of 162 Da corre- sponding to removal of terminal galactose residues were observed for the following structures; mlz 4072.7 to 3912.8; 3780.5/3618.5 to 3459.0; 3414.4 to 3256.5;

4148,113821.5 to 3661.1; 3577.8 to 3418.1 and 3739.4 to 3580.4 (illustrated in Fig. 4, digestion series B). Four structures with terminal sialic acid residues were not observed after the b-galactosidase digestion (indicated ‘Not Obs.’ in Fig. 4). A possible explanation for this might be a partial loss of sialic acid residues during the long incubation time, or that the @-galactosidase contained trace amounts of neuraminidase. Upon N-acetylglucosaminidase digestion [Fig. 3(c)], mass losses of 203 Da corresponding to the removal of N-acetylglucosamine residues were observed for the following structures: m/z 3912.8 to 3708.3; 3459.01 3256.5/3661.1 to 3051.3; 3418.1 to 3213.4 and 3580.4 to 3375.5 (illustrated in Fig. 4, digestion series B). Two

HETEROGENEITY OF INTERFERON-y GLYCANS 1115

structures were incompletely digested and the N - acetylglucosamine residues not completely removed [Fig. 3(c); m/z 3254.5 and 3457.43.

The presence of two high mannose structures was verified by treatment of the native glycopeptide [Fig. 3(a)] with a-mannosidase, which is capable of cleaving off 01-1-2, 1-3 and 1-6 linked mannose residues [Fig. 3(d)]. Mass losses of 162 Da corresponding to the removal of mannose residues were observed for the fol- lowing two structures: m/z 3535.213374.3 to 3213.11 3049.9 (illustrated in the inset in Fig. 4). Mass losses from other structures were not observed.

Overall, the glycan on Asn 97 consisted of a mixture of complex bi- and triantennary structures, high mannose structures and hybrid structures. The major components, according to the peak intensities, were non- and monosialylated biantennary structures and a high mannose structure with five mannose residues. The Asn 97 glycan was not found to be core fucosylated at the innermost N-acetylglucosamine in any of the struc- tures. The molecular masses of two structures indicated that they were fucosylated in the outer antennary struc- ture. Neither of these structures was observed after digestion with /I-galactosidase. This suggests either that the fucose residue is lost during the digestion or that it is positioned on a galactose residue, assuming that /?- galactosidase also cleaves fucosylated /?-galactose resi- dues (indicated with dashed arrows in Fig. 4).

Asparagine 25. The glycan structure at Asn 25 was also elucidated by digestion with two series of exo- glycosidases (illustrated in Fig. 5, spectra not shown) as already described for the Asn 97 glycan. Overall, the glycan at Asn 25 was not as heterogeneous as the glycan on Asn 97 and consisted of a mixture of complex bi-, tri- and tetra-antennary structures and one hybrid structure. High mannose structures were not observed on Asn 25. The major components were mono- and dis- ialylated biantennary structures according to relative signal intensities. The masses determined indicated that the Asn 25 glycan was core fucosylated at the N - acetylglucosamine attached to the asparagine residue in all the structures. This was verified by a digestion with a mixture of endoglycosidase F and N-glycosidase F fol- lowed by a partial digestion with a-fucosidase (Fig. 6).

A partial loss of 146 Da corresponding to the removal of a fucose residue from the reducing-end N - acetylglucosamine was observed. In addition, two struc- tures seemed to be fucosylated in the outer antennary structure. In contrast to the Asn 97 glycan, these fucosy- lated structures were not cleaved by j3-galactosidase.

Heterogeneity caused by mass spectrometric fragmentation

When carbohydrates are analysed by MS, there is often the question of whether some of the observed heter- ogeneity is caused by MS fragmentation. We therefore attempted to examine to what extent the heterogeneity observed was caused by (i) prompt fragmentation and (ii) metastable fragmentation during the mass spectro- metric analysis.

(i) Prompt fragmentation is induced by the laser irra- diation and occurs prior to acceleration of the ions. Structures resulting from prompt fragmentation are not present in the sample before the MS analysis, but are observed in the mass spectra. Consequently, structures deduced as due to prompt fragmentation cannot be digested with exoglycosidases that are specific for the terminal residues, whereas structures present in the sample can. Comparison of the two digestion series of the Asn 97 carbohydrates (Fig. 4, digestion series A and B) shows that each group of native structures were digested with the exoglycosidases and their presence in the sample was thereby verified. When a group of struc- tures upon digestion with an exoglycosidase resulted in one new structure, it cannot be excluded that some of the first set of structures were deduced as a result of prompt fragmentation. In Figs 4 and 5 this is true for any structure within one group (indicated with brackets on the left) in the central column. However, digestion with an alternative exoglycosidase showed that many of these structures were present. In Fig. 4, for instance, it is possible that prompt fragmentation of the first structure (mlz 4364.3) resulted in the appearance of the next three structures (mlz 4073.1, 3781.8 and 3619.7) when only considering the neuraminidase digestion. The structure of mlz 4073.1 was digested with j3-galactosidase, hence it must have been present before the MS analysis. The

Mass [Da] Mass [Da] Figure 6. Linear MALDI mass spectra of the peptide glycosylated at Asn 25 sequentially digested with an endoglycosidase F-N- glycosidase F mixture (A) followed by digestion with a-fucosidase (6). The spectra were obtained using THAP as matrix.

1116 E. M0RTZ ET AL

!!

I?? 3000

Figure 7. Reflected MALDI mass spectrum obtained using 4HCCA as matrix of the peptide glycosylated at Asn 97. Metasta- ble loss of sialic acid residues is detected by comparison with the linear spectrum in Fig. 2(a).

two structures of m/z 3781.8 and 3619.7 were both digested by 8-galactosidase to yield the structure of m/z 3457.5, so at least one of them had to be real, whereas the other might be the result of prompt fragmentation. The first structure of m/z 4364.3 with two terminal sialic acids was not observed after the 8-galactosidase treat- ment, but this cannot be attributed to prompt fragmen- tation of the structure, because then the prompt fragments should appear in the spectra. In general, com- parison of the two digestion series clearly showed that most of the structures were present before the MS analysis.

An alternative method to detect prompt fragmenta- tion is to increase the laser irradiance, which will result in a decreased signal intensity for structures that are fragmenting and an increased signal intensity for the resulting prompt fragments. Increasing the laser irra- diance on the Asn 97 peptide clearly showed an inten- sity increase for the structure of m/z 3781.8 compared with those of m/z 4364.3 and 4073.1 (spectra not shown)

,rI.T ~~~~ , 1 / - - 7 - - - i 7--’

3000 3200 3400 3600 3800 4000 4200 4400 4600

Figure 8. Delayed extraction MALDI mass spectrum obtained using DHB as matrix of the peptide glycosylated at Asn 97. The structures observed in the spectrum are almost identical with the structures observed using 4HCCA and THAP as matrices [Figs 2(a) and 3(a), respectively].

Mass ( d z )

due to prompt fragmentation of the sialic acids. Thus, at least some prompt fragmentation occurred for these structures and possibly also for other structures con- taining sialic acid residues.

(ii) Metastable fragmentation is also induced by the laser irradiance and by collision with other molecules in the gas phase. The metastable fragmentation occurs after the ions have been accelerated and causes broadening of the parent ion peak in the linear spec- trum. Metastable fragments can be detected and their masses determined by comparing the linear and reflec- ted spectra. Comparison of the linear spectrum [Fig. 2(a)] and the reflected spectrum (Fig. 7) of the peptide containing Asn 97 showed that the masses of five sialy- lated structures in the linear spectrum were shifted to the masses of the desialylated structures in the reflected spectrum (m/z 3700.0 to 3417.7; 3868.3 to 3577.4; 4363.9/4073.0 to 3781.3 and 4221.4 to 3927.1). All observed mass shifts were due to metastable loss of sialic acid residues and loss of other residues than sialic acid were not observed.

To investigate the influence of matrix choice on the fragmentation, spectra obtained with 4HCCA and THAP were compared in addition to spectra obtained using delayed extraction with DHB as matrix. 4HCCA is known to produce intense signals from peptides, but also to induce fragmentation of intact proteins and compounds with labile bondsz7 The THAP matrix has previously been used for the analysis of oligonucleotidesz4 which contain labile N-glycosidic linkages. THAP has also been shown to induce less metastable fragmentation than 4HCCA for insulin” and for sulphated peptides (unpublished data). The spectra obtained with 4HCCA and THAP of the peptide containing Asn 97 were very similar [Figs 2(a) and 3(a), respectively] and almost the same structures were observed. Comparison of the linear and reflected spectra obtained in the THAP matrix (reflected spec- trum not shown) showed metastable loss of sialic acid residues, whereas losses of other monosaccharide resi- dues were not observed. Comparison with spectra obtained with the DHB matrix on the Voyager Elite instrument using delayed extraction (Fig. 8) showed almost exactly the same structures as observed for the other two matrices. The resolution was clearly improved in this spectrum, which can probably be attributed to the use of delayed extraction.

DISCUSSION

The MALDI analysis combined with specific glycosi- dase digestion shows that the two glycosylation sites in IFN-), are occupied by glycans that differ in composi- tion and microheterogeneity. These experiments were performed on the mixture of IFN-y forms containing unglycosylated, monoglycosylated and diglycosylated IFN-y molecules. This analysis resulted in a detailed site-specific characterization of the carbohydrate struc- tures present at each glycosylation site in comparison with a previous study performed on the three IFN-y forms after separation by SDS-PAGE, where only the major glycan structure on Asn 25 was identified.I7 Here

HETEROGENEITY OF INTERFERON-I, GLYCANS 1117

we have demonstrated that a mixture or complex glycan structures is attached to Asn 25. All the structures are fucosylated at the reducing-end N- acetylglucosamine. Tri- and tetra-antennary glycans and one hybrid structure are present. No high mannose structures are observed. The glycans on Asn 97 are more heterogeneous, consisting of two high mannose structures and several hybrid structures. In addition, the Asn 97 glycan is not fucosylated at the reducing-end N-acetylglucosamine. These results indicate that the Asn 25 glycan has been further processed in the Golgi complex by the various glycosyl transferases compared with the Asn 97 glycan. A similar preference for pro- cessing of the Asn 25 glycan has also been observed for recombinant IFN-.)I produced in CHO cells and trans- genic animals.1s*29 The differences in the glycan struc- tures at the two glycosylation sites might be attributed to differences in the three-dimensional structure of the protein that might result in reduced accessibility of the various glycosyl transferases to the Asn 97 site.30 This is in agreement with the finding that Asn 25 appears to be glycosylated co-translationally, whereas Asn 97 may be glycosylated post-translationally after the formation of three-dimensional structures, and probably even after the dimerization of IFN-7 has taken place.14

Most of the microheterogeneity observed for the glycans at the two glycosylation sites can be attributed to native structure diversity. A contribution by MS fragmentation to the observed heterogeneity must be considered of minor importance since the presence of most of the structures was verified by digestion with two different series of exoglycosidases. In addition, the same amount of heterogeneity was observed using three different matrices. 4HCCA, THAP and DHB, of which THAP is reported to cause less fragmentation. In fact, the three matrices showed very similar performances for the analysis of the glycosylated IFN-7 peptides. For comparison, the spectra shown in Fig. 2 were all obtained with 4HCCA, those in Fig. 3 were obtained with THAP and that in Fig. 8 with DHB. However, the use of delayed extraction in combination with the DHB matrix showed improved resolution, which might some- times be of importance to resolve carbohydrates that are close in mass. Some of the heterogeneity attributed to different numbers of sialic acid residues may,

however, be caused by prompt fragmentation or by breakage of the labile galactose-sialic acid bonds during the protein purification and proteolytic treatments. Metastable fragmentation was only found to result in loss of sialic acid residues, as previously observed for other glycosylated peptides and protein^.'.'^ Metastable loss of larger fragments of the glycan structure was not investigated in this study, but it has previously been demonstrated that post-source decay analysis of glycans can be used to elucidate partly the glycan Hence we can conclude that the complexity observed in the MALDI mass spectra reflects the complexity present in the glycan structure. We cannot exclude, however, that part of the heterogeneity is caused in the preparation steps prior to the MS analysis.

The use of MALDIiMS combined with specific glyco- sidase digestion has several advantages over traditional methods for the elucidation of glycan structures and for the characterization of glycan microheterogeneity. The advantages include high sensitivity that allows the analysis of small aliquots removed from the digest mixture and further treatment of the remaining sample with other glycosidases; low suppression effects that allow the detection of multiple components in the mixture of oligosaccharide structures; and site-specific information about the glycan structures. However, when the structures of several oligosaccharides are elu- cidated simultaneously, there will always be certain ambiguities in assigning the observed mass shifts to the corresponding structures. The glycosidases used in this study have a broad linkage specificity, i.e. a1-2,1-3 and 1-6 linked mannose residues can all be cleaved by the a-mannosidase from jack bean and these cannot be dis- tinguished. However, once the oligosaccharide sequence has been elucidated, more specific glycosidases could be used to characterize the linkage configurations.

Acknowledgements

Thomas N. Krogh, Henrik Rahbek-Nielsen and Anne K. Kristensen are acknowledged for valuable discussions on the use of glycosidases. We also thank Hannele Tolo (Finish Red Cross Blood Transfusion Service, Helsinki, Finland) for the purified IFN-1. This study was sup- ported by the Protein Engineering Research Centre under the Danish Biotechnology Programme.

REFERENCES

1. R. Kornfeld and S. Kornfeld, Annu. Rev. Biochem. 54. 631

2. T. W. Rademacher, R. B. Parakh and R. A. Dwek, Annu. Rev.

3. C. F. Goochee. M. J. Gramer, D. C. Andersen, J. B. Bahr and

4. A. Varki, Glycobiology 3, 97 (1 993). 5. R. R. Townsend and M. R. Hardy, Glycobiology 1. 139

6. D. C. James, R. B. Freedman, M. Hoare and N. Jenkins, Anal.

7. C. W. Suaon. J. A. O'Neill and J. S. Cottrell, Anal. Biochem.

8. M. C. Huberty. J. E. Vath, W. Yu and S. A. Martin, Anal.

9. B. Stahl, T. Klabunde, H. Witzel, B. Krebs, M. Steup, M. Karas

(7 985).

Biochem. 57.785 (1988).

J. R. Rasmussen, BiolTechnology 9,1347 (1 991 ).

(1991).

Biochem. 222, 31 5 (1 994).

218,34 (1994).

Chem. 65,2791 (1993).

and F. Hillenkamp. Eur. J. Biochem. 220,321 (1 994).

10. S. A. Carr, M. J. Huddleston and M. F. Bean, Protein Sci. 2, 183 (1 993).

11. G. D. Roberts, W. P. Johnson, S. Burman, K. R. Anumula and S. A. Carr, Anal. Chem. 67, 361 3 (1 995).

12. C. J. Edge, T. W. Rademacher, M. R. Wormald, R. B. Parekh, T. D. Butters, D. R. Wing and R. A. Dwek, Roc. Natl. Acad. Sci. USA, 89,6338 (1992).

13. J. H. G. M . Mutsaers, J. P. Kamerling, R. Devos, Y. Guisez, W. Fiers and J. F. G. Vliegenthart, Eur. J. Biochem. 156, 651 (1 986).

14. T. Sareneva, J. Pirhonen, K. Cantell, N. Kalkkinen and I . Julk- unen. Biochem. J. 303,831 (1 994).

15. D. C. James, R. B. Freedman, M. Hoare, 0. W. Ogonah, B. C. Rooney, 0. A. Larinov, V. N. Bobrovolsky, 0. V. Lagutin and N. Jenkins, BiolTechnology, 3, 592 (1 995).

16. E. Rinderknecht. B. H. O'Connor and H. Rodriguez, J. Bid. Chem ,259,6790 (1 984).

1118 E. M0RTZ ET AL.

17. E. Mertz, T. Sareneva, S. Haebel, I. Julkunen and P. Roep-

18. T. Sareneva, J. Pirhonen, K. Cantell and I. Julkunen, Biochem. storff, Electrophoresis 17,925 (1 996).

J. 308,9 (1 995). 19. S. Yarnamoto, S. Hase, H. Yamauchi,

Ikenaka, J. Biochem. 105,1034 (1989). 20. N. Jenkins, Biochem. SOC. Trans., 23, 17 21. K. Cantell, S. Hiorvonen and H.-L.

Enzymol., 119, 54 (1986).

T. Tanimoto and T.

(1995). Kau pinen, Methods

22. H.-L. Kauppinen, B. Bang, J. Eronen, R. Majuri, G. Myllyla, H. TOIO, S. Hirvonen and K. Cantell, in The Biology of the Interferon System 1985, edited by W. E. Stewart, II and H. Schellekens, pp. 221 -227. Elsevier, Amsterdam (1 986).

23. 0. Vorrn, P. Roepstorff and M. Mann, Anal. Chem. 66, 3281 (1 994).

24. U. Pieles, Zurcher, M. Schar and H. E. Moser, Nucleic Acids Res. 21,3191 (1993).

25. 0. Vorrn and M. Mann, J. Am. SOC. Mass Spectrom. 5, 955 (1 994).

26. M. L. Vestal, P. Juhasz and S. A. Martin, Rapid Common. Mass Spectrom. 9, 1 044 (1 995).

27. M. Karas, U. Bahr, K. Strupat, F. Hillenkamp, A. Tsarbopoulus and B. Promanik, Anal. Chem. 67,675 (1 995).

28. 0. Vorrn, PhD Thesis, Odense University (1995). 29. N. Jenkins, Biochem. SOC. Trans., 23, 171 (1995). 30. S. E. Ealick, W. J. Cook, S. Vijay-Kumar, M. Carson, T.-L.

Nagabhushan, P. P. Trotta and C. E. Bugg, Science 252, 698

31. G. Talbo and M. Mann, Rapid Commun. Mass Spectrom. 10, (1991).

100 (1 996).