J. Agric. Food Chem. 1999, 47, 4549-4556
`
`4549
`
`Use of Mass Spectrometry To Rapidly Characterize the
`Heterogeneity of Bovine oL—Lactalbumin
`
`Department of Product Technology, NIZO Food Research, P.O. Box 20, 6710 BA Ede, The Netherlands
`
`Charles J . Slangen and Servaas Visser*
`
`From a bovine whey protein fraction the nonglycosylated and glycosylated 0L—lactalbumin fractions
`were isolated by gel—permeation chromatography followed by reversed—phase high—performance liquid
`chromatography. Both fractions were studied by electrospray—ionization mass spectrometry (ESI—
`MS). For the nonglycosylated fraction, a mass of 14 178 Da was found, which was in accordance
`with the known amino acid sequence of the protein. The glycosylated fraction appeared to be a
`mixture of components with mass values ranging from ca. 15 840 to 16 690 Da. Given the published
`carbohydrate composition of the whole glyco—0L—lactalbumin fraction, these masses could be matched
`to those of 14 differently glycosylated forms of 0L—lactalbumin. Further support for these forms was
`obtained from the results of a separate mass spectrometric analysis of the mixture of oligosaccharides
`released from the protein by treatment with peptide—1\/4-(Z\flacetyl—f3—glucosaminyl)asparagine amidase
`F. ESI—MS was found to be a powerful tool to gain insight into the composition of the complex mixture
`of glycoforms of 0L—lactalbumin without the need of further purification of these forms or of the
`oligosaccharides released thereof.
`
`0L—Lacta]bL1m1'n,' (de—)g]yc0sy]at1'0n,' s1'ze—exc]L1s1'0n chromatography; reversed—phase HPL C;
`Keywords:
`H7855 Spl-Z‘CIfI”0IH6‘II”_y
`
`INTRODUCTION
`
`0L—Lactalbumin, a major whey protein in milk of
`various species, plays an important role in the biosyn—
`thesis of lactose (Brodbeck and Ebner, 1966; Ebner et
`al., 1966; Brew and Grobler, 1992). On the basis of the
`most recent sequence data (Wang et al., 1989), the
`molecular mass of bovine 0L—lactalbumin is 14 178 Da.
`
`However, in fresh, nonprocessed milk, a small part of
`the native protein occurs in the glycosylated form (glyco—
`0L—lactalbumin)
`(Barman, 1970). By monosaccharide
`analysis of glyco—0L—lactalbumin, varying amounts of
`N—acetylglucosamine (GlcNAc), N—acetylgalactosamine
`(GalNAc), mannose (l\/Ian), galactose (Gal), fucose (Fuc),
`and N—acetylneuraminic acid (NeuAc) were found, the
`quantities of Gal, Fuc, and NeuAc being relatively low
`(Barman, 1970; Hindle and Wheelock, 1971). In bovine
`0L—lactalbumin, Asn—45 was reported to be the single
`point of attachment of carbohydrate groups (Hopper and
`McKenzie, 1973; Hill and Brew, 1975), although accord-
`ing to the usual consensus sequence for N—linked
`carbohydrate chains (Asn—X—Thr/Ser) one more potential
`glycosylation site is present at Asn—74. It is noteworthy
`that the carbohydrate moiety does not seem to be
`involved in the role of 0L—lactalbumin in lactose biosyn—
`thesis, since the glycosylated and nonglycosylated forms
`are equally active as lactose synthase specifier protein
`(Barman, 1970).
`
`Hopper and McKenzie (1973) found that glyco— and
`nonglyco—0L—lactalbumin forms could be separated by
`Sephadex G—75 chromatography. Further purification
`by DEAE ion—exchange chromatography resulted in two
`electrophoretically distinguishable glyco—0L—lactalbumin
`
`* To whom correspondence should be addressed (fax +31
`318 650400; e—mail svisser@nizo.nl).
`
`fractions (S1 and S2) and a main and minor component
`(M and F) which both were nonglycosylated.
`No further data on the structure of the carbohydrate
`moiety of glyco—0L—lactalbumin were reported until the
`publication of a symposium abstract by Tilley et al.
`(1991). In this note not only were the carbohydrate
`composition and single glycosylation site of glyco—0L—
`lactalbumin confirmed, but also the oligosaccharide
`structure of a main glycoform of 0L—lactalbumin was
`shown.
`
`For the determination of one or more glycosylation
`sites and the detailed structure of attached carbohy-
`drate chains it is usual
`to proteolytically digest a
`glycoprotein and fractionate the digest to fragments
`each containing only one glycosylated position. The
`glycopeptides obtained or the oligosaccharides released
`thereof are purified and then analyzed by, for instance,
`NMR spectrometry (Vliegenthart et al., 1983, 1991; van
`Halbeek, 1989; Vliegenthart and Montreuil, 1995).
`Recent developments in mass spectrometric techniques
`have opened new ways into the study of glycoproteins
`(Vliegenthart and Montreuil, 1995; Rademaker and
`Thomas—Oates, 1996; Harvey, 1996; Settineri and Bur-
`lingame, 1996; Green et al., 1996).
`In the present paper we describe the isolation and
`purification of the nonglycosylated and glycosylated
`bovine 0L—lactalbumin fractions as well as their charac-
`
`terization by electrospray—ionization mass spectrometry
`(ESI—MS). On the basis of our results and the published
`carbohydrate composition of total glyco—0L—lactalbumin,
`the compositions of 14 glycoforms of 0L—lactalbumin are
`postulated. The presence of these glycoforms is sup-
`ported by ESI—MS results obtained with the whole
`mixture of oligosaccharides enzymatically released from
`the protein chain by peptide—1\/4-(N—acetyl—fi—glucosami—
`nyl)asparagine amidase (PNGase F).
`
`© 1999 American Chemical Society
`10.1021/jf990212j CCC: $18.00
`Published on Web 10/28/1999
`
`Amgen Exhibit 2010
`Apotex Inc. et al. V. Amgen Inc. et al., IPR2016-01542
`Page 1
`
`

`
`4550
`
`J. Agric. Food Chem., Vol. 47, No. 11, 1999
`
`Slangen and Visser
`
`CB
`PAS
`/4? /4}
`
`(+3
`
`A230
`
`X
`
`jT
`
`fl if
`
`DITWPEI Y ]]1TWP]E Y
`
` 0
`
`100‘
`
`200
`
`300
`
`Figure 1. Separation of a whey protein fraction (1 g) on a 140 X 2.6 cm Sephadex G—75 Superfine column (for experimental
`conditions, see text). The rounding of peak summits is indicative of “overconcentration" in the photocell. Material from peaks III
`(fi—lactoglobulin), IV (glyco—oL—lactalbumin) and V (nonglyco—oL—lactalbumin) was analyzed by native PAGE followed by staining
`with periodic acid—Schiff reagent (PAS) or Coomassie Blue G—250 (CB), as illustrated by the inset. TWP = total whey protein.
`
`Elution volume (mL]
`
`MATERIALS AND METHODS
`
`Materials. A bovine whey protein fraction, oL—lactalbumin—
`enriched byproduct (“fraction P2") of the classical /j’—lactoglo—
`bulin purification according to Aschaffenburg and Drewry
`(1957), was used as starting material for the isolation of the
`main (nonglycosylated) oL—lactalbumin fraction and of the minor
`fraction containing glyco—oL—lactalbumin. For comparative pur-
`poses, an oL—lactalbumin fraction prepared on a technical scale
`from concentrated cheese whey by ion—exchange chromatog-
`raphy on DEAE—Sepharose (Schmidt and van Markwijk, 1993)
`was employed.
`PNGase F was purchased from Boehringer—Mannheim,
`Germany. Trifluoroacetic anhydride was a product of Sigma
`(St. Louis, MO). All other chemicals were of analytical grade.
`Chromatographic Isolation and Gel—Electrophoretic
`Characterization. The isolation of oL—lactalbumin was per-
`formed essentially as described by Davies (1974) with some
`modifications. The oL—lactalbumin—enriched whey protein frac-
`tion (1 g dissolved in 3 mL of elution buffer, 0.01 M
`imidazole-HCl/0.2 M NaCl/0.02% NaN3, pH 6.65) was carefully
`loaded onto the top of a 140 X 2.6 cm Sephadex G—75 Superfine
`column (Pharmacia, Uppsala, Sweden) and eluted with elution
`buffer at room temperature and at a flow rate of 30 mL h’1.
`Absorbance was continuously measured at 280 nm using a
`Uvicord absorptiometer, type 8303A (LKB, Bromma, Sweden).
`Peak fractions were collected, dialyzed against distilled water
`at 4 °C, and freeze—dried.
`Polyacrylamide gel electrophoresis (PAGE) at pH 8.6 was
`carried out in a vertical slab gel apparatus (E—C Corporation,
`St. Petersburg, FL) as described by Davies (1974). Staining
`for proteins was with Coomassie Blue G—250 and for glyco-
`proteins with periodic acid—Schiff reagent (PAS) (Kapitany and
`Zebrowski, 1973).
`Further purification of oL—lactalbumin fractions was per-
`formed by semipreparative reversed—phase (RP)—HPLC using
`a water—acetonitrile gradient elution, essentially as described
`elsewhere (Visser et al., 1991).
`Enzymatic Deglycosylation of oL—Lactalbumin and
`Acetylation of Released Oligosaccharides. Deglycosyla-
`tion of glyco—oL—lactalbumin was carried out with PNGase F as
`described by Rademaker and Thomas—Oates (1996). The gly-
`coprotein (0.33 mg dissolved in 200 ,uL of 50 mM ammonium
`bicarbonate buffer, pH 8.4, containing 1 drop of toluene) was
`incubated with 5 ,uL (1 unit) of PNGase F solution at 37 °C
`for 16 h. The resulting mixture of deglycosylated oL—lactalbumin
`and released oligosaccharides was freeze—dried, redissolved in
`500 ,uL of 5% acetic acid, and separated on a Sep—Pak Plus
`
`C18 cartridge (Waters, Milford, MA) which had been prewetted
`with methanol and equilibrated with 5% acetic acid. The
`oligosaccharides were eluted with 5% acetic acid in the first
`eluate. The vacuum—dried (SpeedVac vacuum centrifuge, Sa-
`vant Instruments, Farmingdale, NY) eluate was peracetyl—
`ated using a mixture of trifluoroacetic anhydride and acetic
`acid (together forming a mixed anhydride). After another
`evaporation step, the dried product was dissolved in dichlo-
`romethane and washed several times with distilled water
`(Rademaker and Thomas—Oates, 1996). The vacuum—dried
`mixture of oligosaccharides was then ready for mass spectro-
`metric analysis. The deglycosylated glyco—oL—lactalbumin was
`obtained from a second eluate fraction (20—40% 1—propanol
`in 5% acetic acid) during the Sep—Pak Plus C18 separation.
`Mass Spectrometry. For the analysis of the oL—lactalbumin
`fractions, ESI mass spectra were obtained with a Quattro II
`triple quadrupole instrument (Micromass, Cheshire, U.K.).
`Samples were dissolved in 50% acetonitrile, 0.3% formic acid,
`and analyzed by infusion with a syringe infusion pump, type
`22 (Harvard Apparatus, South Natick, MA), of the sample
`solution in 50% acetonitrile at 4 ,uL min‘1 through the
`electrospray interface. Nitrogen was used as a nebulizing and
`drying gas. The capillary was held at 3.9 kV; the cone voltage
`was 40 V. The acetylated carbohydrate fraction was dissolved
`in 50% methanol and 0.3% formic acid and analyzed by
`infusion as described above with the capillary held at 3.5 kV
`and the cone voltage at 30 V. Mass calibration was performed
`by multiple—ion monitoring of a NaI/CsI mixture. About 10-
`15 spectra were averaged to obtain an adequate signal—to—noise
`ratio. The raw mass spectral data were processed and trans-
`formed with the Masslynx software version 2.2 (Micromass,
`Cheshire, U.K.) on a Windows NT workstation.
`
`RESULTS AND DISCUSSION
`
`Chromatographic Isolation and Gel-Electro-
`phoretic Characterization of ot-Lactalbumin Frac-
`tions. The fractionation of the whey protein fraction on
`a Sephadex G—75 Superfine column is illustrated by
`Figure 1 (numbering of peaks according to Armstrong
`et al., 1970), with the PAGE gel patterns of isolated
`fractions III (ffilactoglobulin), IV (mainly glyc0—oL—lact—
`albumin), and V (n0nglyco—oL—lactalbumin) shown in the
`inset of that figure. Fraction I (pink) and II (yellowish),
`both not shown on the gel, included minor whey proteins
`such as lactoferrin, 3/—globulins, and serum albumin
`(Davies, 1974). Particularly when focusing on a satisfac-
`
`Page 2
`
`

`
`MS Study of ot-Lactalbumin Heterogeneity
`
`J. Agric. Food Chem., Vol. 47, No. 11, 1999
`
`4551
`
`
`
`Absorbance(220nm)
`
`0
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`Figure 2. Reversed—phase HPLC patterns of (a) peak component IV of Figure 1, (b) peak component V of Figure 1, and (c) a
`sample of oL—lactalbumin prepared by DEAE—Sepharose chromatography on a technical scale. Peak material was collected and
`used for mass spectrometric analysis.
`
`Elution time (min)
`
`tory separation between glyco—oL—lactalbumin and the
`other fractions, we obtained better results with Sepha—
`dex G—75 than by using G—100 material. The latter had
`been used in the method of Davies (1974), who sepa-
`rated ca. 50 mg amounts of whey protein on a much
`shorter Sephadex G—100 column, collecting fractions IV
`and V together. Starting from 1 g of whey protein
`fraction, we obtained ca. 70 and 380 mg of dry material
`after dialysis and freeze—drying of fractions IV and V,
`respectively. The two electrophoretic glycoforms (PAS-
`positive) found in fraction IV (Figure 1) are the same
`as those reported in the literature (Barman, 1970;
`Hopper and McKenzie, 1973; Davies, 1974; Armstrong
`et al., 1970; Baumy and Fauquant, 1989) and represent
`the S1 and S2 fractions found by Hopper and McKenzie
`(1973)
`to include NeuAc—containing and NeuAc—free
`components, respectively.
`RP—HPLC patterns of fractions IV and V (Figure 1)
`are seen in Figure 2 (traces a and b) together with, for
`comparison, the pattern of an oL—lactalbumin preparation
`produced from cheese whey by DEAE—Sepharose chro-
`matography (trace c)
`(Schmidt and van Markwijk,
`1993). Glyco—oL—lactalbumin (trace a) still contained a
`major and a minor peak, in an area ratio of about 70:
`30. Assuming that the minor peak represents a chemi-
`cally induced oL—lactalbumin derivative (see next sec-
`tion), we may conclude that, on the basis of the above
`yields of fractions IV and V, ca. 10% (m/m) of oL—lactal—
`bumin in our starting material existed in the glycosyl—
`ated form. This is a significantly higher percentage than
`the 3% obtained by Hopper and McKenzie (1973) when
`separating total whey protein via a similar gel—filtration
`technique. Our percentage only slightly exceeds the 7%
`content reported by Baumy and Fauquant (1989), who
`used Sephadex G—50 chromatography and started from
`an industrial oL—lactalbumin preparation. The minor
`component eluting just before the main one in trace 0
`of Figure 2 is different from the minor component in
`
`trace a. It had an elution time corresponding to that of
`the last part of the main peak in trace a and, as seen in
`the next section, contained only part of the glycoforms
`of oL—lactalbumin. Several peak fractions of the RP-
`HPLC separations were collected and dried by vacuum
`centrifugation (SpeedVac) before use in mass spectro-
`metric analyses.
`Mass Spectrometry of (1-Lactalbumin Compo-
`nents and Released Oligosaccharides. Mass spectra
`and deconvoluted spectra of the main RP—HPLC com-
`ponents representing glyco— and nonglyco—oL—lactalbumin
`(see Figure 2) are depicted in Figures 3-5. It was found
`that nonglyco—oL—lactalbumin had a mass of 14 178.8 Da
`(Figure 3), which was in agreement with the theoretical
`value of 14 178 Da (Wang et al., 1989). The minor peak
`following the main one in the RP—HPLC pattern of
`Figure 2, trace 21, cannot be ascribed to a natural glyco—
`oL—lactalbumin form, as the corresponding mass (14 503
`Da) was too low to account for any oligosaccharide chain
`attached to oL—lactalbumin (result not shown). We have
`experimental evidence now that this component results
`from a chemical
`lactosylation process taking place
`during heat processing of milk (Slangen and Visser,
`unpublished). Recently, such components were also
`reported to occur in isolated ffilactoglobulin (Burr et al.,
`1996; Léonil et al., 1997). They can be considered as
`early Maillard products. A similar product, isolated from
`human blood serum, was found to be a reaction product
`of serum albumin and blood glucose (Bunk, 1997).
`The mass spectrum of glyco—oL—lactalbumin (Figure 4)
`was fairly complicated and contained, after deconvolu-
`tion, at least 15 discrete peaks (Figure 5). Given the
`presence of GlcNAc, GalNAc, Man, Gal, Fuc, and NeuAc
`in glyco—oL—lactalbumin (Barman, 1970; Hindle and
`Wheelock, 1971) and the known core structure of
`N—linked carbohydrates, we were able to match the
`masses of the various components shown in Figure 5 to
`those of 14 glycoforms of oL—lactalbumin (Table 1).
`
`Page 3
`
`

`
`4552
`
`J. Agric. Food Chem., Vol. 47, N0. 11, 1999
`
`Slangen and Visser
`
`m0_
`
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`
`Figure 3. Electrospray mass spectrum of purified nonglyco—oL—lactalbumin (main peak in Figure 2, trace b). Charge numbers of
`the molecular—ion clusters are also indicated, with a maximum mass intensity at nine charges (A9). The inset shows the deconvoluted
`spectrum.
`
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`
`Figure 4. Electrospray mass spectrum of purified glyco—o1—lactalbumin (main peak in Figure 2, trace a). AX, BX, and CX denote
`components of the three main envelopes of the spectrum.
`
`Our results indicate that 8 of the 14 proposed glyco-
`forms concern sialylated oligossacharide chains. This
`distinction between sialylated and nonsialylated oli-
`
`gosaccharides would account for the presence of the two
`PAS—positive bands in the PAGE—pattern of glyco—0L—
`lactalbumin (Figure 1) (Hopper and McKenzie, 1973).
`
`Page 4
`
`

`
`MS Study of 01-Lactalbumin Heterogeneity
`
`J. Agric. Food Chem., Vol. 47, N0. 11, 1999 4553
`
`16030
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`Mass (Da)
`
`Figure 5. Deconvoluted mass spectrum of purified glyco—oL—lactalbumin (see Figure 4). The mass components numbered 1-14
`are referred to in Table 1.
`
`Table 1. Mass Data and Possible Monosaccharide
`Composition for Glycosylated Forms of Bovine
`oL—Lactalbumin""
`
`structure published so far (Tilley et al., 1991) and
`reported to represent the major glycoform
`
`Fuc
`
`GalNAc- G1cNAc-Man \
`Ga1NAc-GlcNAc-Man
`
`Man-G1cNAc-GlcNAc-Asn“
`
`corresponds with that of component 5 in Figure 5 and
`Table 1; this component was also found in the present
`study to give a relatively high response (Figure 5). It is
`likely to assume that the other glycoforms shown in
`Table 1 represent, by analogy with recently published
`milk glycoprotein structures (Coddeville et al., 1992;
`Sato et al., 1993; Girardet et al., 1995), closely similar
`structures, being either fucosylated or nonfucosylated,
`sialylated or nonsialylated, and having in a number of
`cases Gal instead of GalNAc. This would also be in line
`
`with the relatively small amounts of Gal, Fuc, and
`NeuAc that were earlier reported to occur in glyco—oL—
`lactalbumin (Barman, 1970; Hindle and Wheelock,
`1971). The occurrence of GalNAc, earlier considered as
`rather unusual in N—linked oligosaccharides (Tilley et
`al., 1991; Montreuil, 1980), has recently been reported
`for several glycoproteins of this category (Tilley et al.,
`1991; Sato et al., 1993; Girardet and Linden, 1996). It
`was suggested to be controlled by an N—acetylgalac—
`tosaminyltransferase acting in competition with a ga-
`lactosyltransferase on common, terminal—GlcNAc—con—
`taining substrate sites (Girardet and Linden, 1996;
`Neeleman et al., 1994).
`Molecular masses of glyco—oL—lactalbumin reported in
`the literature were calculated from amino acid and
`
`Page 5
`
`monosaccharide
`composition
`based on
`measured mass
`HexN Fuc Hex NeuAc
`
`\lC3\l\lO><.I1>-l>O)<.J‘10><.I1>J>O><.J‘1
`
`papa:3papapapa:3:3papapac3c3
`
`waLnaa6;L06:LnLoaaLoJaLnLowa
`
`papapa:3papapapapa:3:3:3c3c3
`
`calcd
`mass
`
`calcd residue
`mass of glycan
`15842.62 1664.54 (2691.45)
`15883.68 1705.60 (2690.47)
`15947.70 1769.63 (2922.66)
`15988.76 1810.69 (2921.67)
`16029.82 1851.74 (2920.69)
`16133.88 1955.80 (3108.82)
`16174.94 1996.85 (3107.84)
`16238.96 2060.89 (3340.03)
`16280.02 2101.95 (3339.04
`16321.08 2143.00 (3338.06
`16395.14 2217.08 (3496.21
`16540.28 2362.19 (3683.36
`16645.36 2467.28 (3914.56
`16686.42 2508.34 (3913.58
`
`))))))
`
`measured
`mass
`
`5o
`
`15841.1
`15882.8
`15946.7
`15987.5
`16029.5
`16133.0
`16175.3
`16240.2
`16280.3
`16320.2
`16394.3
`16539.5
`16646.6
`16685.3
`
`£DO0\lG><.H>J>L«>[\.)>d
`
`10
`11
`12
`13
`14
`
`5 Calculated masses of the glycoprotein components are based
`on the proposed monosaccharide composition in combination with
`a calculated average mass of 14178.06 for nonglycosylated 0L—lac—
`talbumin (Wang et al., 1989). Calculated average masses of the
`released and fully acetylated glycan moieties, including 102.09 for
`terminal—group acetylation, are indicated in parentheses (experi-
`mental values to be derived from Figure 6). HexN = GalNAc/
`GlcNAc: average residue mass 203.20 (acetylated, 287.27). Fuc:
`average residue mass 146.14 (acetylated, 230.22). Hex = Gal/Man:
`average residue mass 162.14 (acetylated, 288.23). NeuAc: average
`residue mass 291.26 (acetylated, 417.37).
`
`It should be borne in mind that the mass spectrometric
`results do not necessarily reflect the various glyco—oL—
`lactalbumin forms in a quantitative manner. On the
`other hand, the mass of the only glyco—oL—lactalbumin
`
`

`
`4554
`
`J. Agric. Food Chem., Vol. 47, No. 11, 1999
`
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`Figure 6. Deconvoluted mass spectrum of enzymatically released and peracetylated oligosaccharides originating from glyco—oL—
`lactalbumin. Number of acetyl groups missing as a result of incomplete derivatization are indicated by X in
`where (y)
`corresponds to the numbering of components as used in Table 1 and Figure 5. Several oligosaccharides could not be distinguished
`because of a similar mass of their acetylated forms.
`
`Mass (Da)
`
`carbohydrate analyses or from retention behavior dur-
`ing gel—permeation chromatography. Values obtained
`were between 16 300 and 16 800 Da (Barman, 1970;
`Hopper and McKenzie, 1973; Baumy and Fauquant,
`1989), which is within the mass range of the various
`individual components found in the present study (Table
`1).
`The glycoforms postulated in Table 1 were supported
`by mass spectrometric analysis of the oligosaccharide
`fraction obtained after deglycosylation of glyco—oL—lact—
`albumin with PNGase F and subsequent peracetylation.
`No distinction could be made between several acetylated
`oligosaccharides of similar mass. Acetylation was car-
`ried out in order to allow the subsequent extraction of
`derivatized oligosaccharides into the organic phase,
`which offers a more effective separation from contami-
`nants (e.g., salts) and therefore enhances the sensitivity
`of detection during mass spectrometric analysis (Rade—
`maker and Thomas—Oates, 1996). The molecular mass
`of the deglycosylated glyco—oL—lactalbumin was found to
`be 14 178.5 Da (calculated: 14 179 Da for oL—lactalbumin
`with Asn—45 converted to Asp as a result of the degly-
`cosylation procedure). The deconvoluted mass spectrum
`of the released and acetylated oligosaccharides is shown
`in Figure 6. Taking into account the occurrence of 2-4
`positive charges per molecule, a spectrum was obtained
`in which nearly all peaks could be assigned by assuming
`for each oligosaccharide moiety 0-3 acetyl residues less
`as a result of incomplete acetylation. Masses correlated
`very well with the compositions based solely on masses
`of total glycoprotein molecules, as postulated in Table
`1. On the basis of peak intensities, the main oligosac-
`
`charides as derived from Figure 6 corresponded to the
`main glycoprotein components deduced from Figure 5.
`The fact that complete deglycosylation could be achieved
`with nondenatured glyco—oL—lactalbumin indicates that
`the protein—glycan linkage is easily accessible to the
`enzyme. As recently reported (Pike et al., 1996), Asn—
`45 is indeed located in a solvent—exposed loop of the
`(rather compact) oL—lactalbumin molecule.
`
`For comparison, we have also analyzed the minor
`peak preceding the major one in trace 0 of Figure 2. It
`represented the glycoform of an oL—lactalbumin sample
`prepared in a quite different way (see Materials and
`Methods) without the specific intention of isolating all
`the glycosylated forms. Indeed, this glyco—oL—lactalbumin
`preparation appeared to contain only part of the
`glycoforms shown in Table 1 (mainly components 1, 6,
`7, 9, and 10; result not shown). Therefore, it is likely
`that the number and ratio of glyco—oL—lactalbumin forms
`found depend on the material used and, more particu-
`larly, on the mode of its isolation and purification.
`
`it may be concluded that our mass
`In summary,
`spectrometric analysis of the nonglycosylated fraction
`of bovine oL—lactalbumin has led to a mass value which
`
`is in perfect agreement with the amino acid sequence
`known for this protein (Wang et al., 1989). The glyco-
`oL—lactalbumin fraction, although showing only two
`electrophoretic components on PAGE, exhibits a high
`degree of heterogeneity when analyzed by ESI—MS. For
`the most part this carbohydrate heterogeneity probably
`concerns closely similar structures, either fucosylated
`
`Page 6
`
`

`
`MS Study of ot-Lactalbumin Heterogeneity
`
`J. Agric. Food Chem., Vol. 47, No. 11, 1999 4555
`
`or nonfucosylated and sialylated or nonsialylated,
`whereas in quite a number of cases Gal is replaced by
`GalNAc.
`
`While data such as overall carbohydrate composition
`and single, N—linked glycosylation site were already
`known from the literature, our study demonstrates that
`mass spectrometry is a powerful tool to gain further
`insight
`into the structural composition of glyco—oL—
`lactalbumin. In our case this could be achieved without
`
`any further purification of the glycoprotein fraction or
`of the oligosaccharides released thereof. In case any
`ambiguity remained concerning the composition of some
`glycan residue at Asn—45 after ESI—MS of the total
`glycoprotein fraction, the independent second approach
`of ESI—MS analysis of the released and peracetylated
`oligosaccharide fraction provided enough evidence to
`make the appropriate choice. For instance, the mass
`found for glycoprotein component 13 of Table 1 (16 646.6
`Da) could also be explained by the glycan composition
`of component 10 plus one lactose residue resulting from
`lactosylation at some lysine residue (calculated mass
`16 645.1 Da). However, the latter assumption becomes
`unlikely by considering the results of the second ap-
`proach of ESI—MS analysis of the oligosaccharide frac-
`tion, i.e., the masses of 3912 and 3871 Da assigned to
`the acetylated glycan of component 13 (Figure 6).
`Moreover, no mass peaks were found which could
`represent any other mixed oligosaccharide/lactose-
`containing component.
`The biological function of the glyco—oL—lactalbumin
`fraction still remains unclear. It has been suggested
`(Barman, 1970) that in the Golgi region of the mammary
`gland cells oL—lactalbumin is wholly secreted as a glyco-
`protein and subsequently degraded by milk glycoside
`hydrolases. If this would be the case, then the biological
`significance of the glycan moiety in oL—lactalbumin might
`be limited to its role in the labeling of the protein for
`export from the cell. However, other biological functions
`cannot be excluded and may be a subject for further
`study.
`
`ABBREVIATIONS USED
`
`ESI—MS, electrospray—ionization mass spectrometry;
`PAS, periodic acid—Schiff reagent; PNGase F, peptide-
`l\/4—(l\flacetyl—fi—glucosaminyl)asparagine amidase F; RP-
`HPLC, reversed—phase high—performance chromatogra-
`phy-
`
`ACKNOWLEDGMENT
`
`We thank Peter van Rooijen for the isolation of glyco-
`oL—lactalbumin and Janneke Kromkamp for providing us
`with an oL—lactalbumin sample prepared on a technical
`scale. We are very grateful to Dr. Jane Thomas—Oates
`(University of Manchester, U.K.) for fruitful discussions
`and helpful suggestions.
`
`Supporting Information Available: Possible glycan
`structures at Asn—45 of bovine glyco—oL—lactalbumin components
`1- 14 based on the results presented in Figure 5 and Table 1
`of this paper.
`
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`Page 7
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`4556
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`J. Agric. Food Chem., Vol. 47, No. 11, 1999
`
`cercariae of the schistosome Trichobilharzia ocellata. Ca-
`
`talysis of a key ste