J. Agric. Food Chem. 1999, 47, 4549-4556
`
`4549
`
`Use of Mass Spectrometry To Rapidly Characterize the
`Heterogeneity of Bovine a-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 wheyprotein fraction the nonglycosylated and glycosylated a-lactalbumin fractions
`were isolated by gel-permeation chromatography followed by reversed-phase high-performanceliquid
`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-a-lactalbumin fraction, these masses could be matched
`to those of 14 differently glycosylated forms of a-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-M*-(N-acetyl-6-glucosaminyl)asparagine amidase
`F. ESI-MSwas found to be a powerful tool to gain insight into the composition of the complex mixture
`of glycoforms of a-lactalbumin without the need of further purification of these forms or of the
`oligosaccharides released thereof.
`
`Keywords: a-Lactalbumin; (de-)glycosylation; size-exclusion chromatography; reversed-phase HPLC;
`Mass spectrometry
`
`INTRODUCTION
`
`a-Lactalbumin, a major whey protein in milk of
`various species, plays an important role in the biosyn-
`thesis of lactose (Brodbeck and Ebner, 1966; Ebneret
`al., 1966; Brew and Grobler, 1992). On the basis of the
`most recent sequence data (Wang et al., 1989), the
`molecular mass of bovine a-lactalbumin is 14 178 Da.
`However, in fresh, nonprocessed milk, a small part of
`the native protein occurs in the glycosylated form (glyco-
`a-lactalbumin)
`(Barman, 1970). By monosaccharide
`analysis of glyco-a-lactalbumin, varying amounts of
`N-acetylglucosamine (GlcNAc), N-acetylgalactosamine
`(GalNAc), mannose (Man), galactose (Gal), fucose (Fuc),
`and N-acetylneuraminic acid (NeuAc) were found, the
`quantities of Gal, Fuc, and NeuAcbeingrelatively low
`(Barman, 1970; Hindle and Wheelock, 1971). In bovine
`a-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 a-lactalbumin in lactose biosyn-
`thesis, since the glycosylated and nonglycosylated forms
`are equally active as lactose synthasespecifier protein
`(Barman, 1970).
`Hopper and McKenzie (1973) found that glyco- and
`nonglyco-a-lactalbumin forms could be separated by
`Sephadex G-75 chromatography. Further purification
`by DEAEion-exchange chromatography resulted in two
`electrophoretically distinguishable glyco-a-lactalbumin
`
`* To whom correspondence should be addressed (fax +31
`318 650400; e-mail svisser@nizo.nl).
`
`fractions (S; and Sz) 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-a-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-a-
`lactalbumin confirmed, but also the oligosaccharide
`structure of a main glycoform of a-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 analyzedby,for instance,
`NMRspectrometry (Vliegenthartet al., 1983, 1991; van
`Halbeek, 1989; Vliegenthart and Montreuil, 1995).
`Recent developments in mass spectrometric techniques
`have opened new waysinto 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 a-lactalbumin fractions as well as their charac-
`terization by electrospray-ionization mass spectrometry
`(ESI-MS). On thebasis of our results and the published
`carbohydrate composition of total glyco-a-lactalbumin,
`the compositions of 14 glycoformsof a-lactalbumin are
`postulated. The presence of these glycoforms is sup-
`ported by ESI-MS results obtained with the whole
`mixtureof oligosaccharides enzymatically released from
`the protein chain by peptide-NM"-(N-acetyl-8-glucosami-
`nyl)asparagine amidase (PNGase F).
`
`© 1999 American Chemical Society
`10.1021/f990212) 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
`oO ee
`
`®©
`
`Asgo
`
`3°.
`
`a
`
`—_
`teal
`
` 0
`
`200
`
`300
`
`WTwe ivy ¥ MTWPIYY ¥
`
`
`100 -
`
`Elution volume (mL)
`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 peaksIII
`(6-lactoglobulin), IV (glyco-c-lactalbumin) and V (nonglyco-a-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 wheyprotein.
`
`MATERIALS AND METHODS
`
`Materials. A bovine whey protein fraction, a-lactalbumin-
`enriched byproduct (“fraction P2”) of the classical f-lactoglo-
`bulin purification according to Aschaffenburg and Drewry
`(1957), was used as starting material for the isolation of the
`main (nonglycosylated) o-lactalbumin fraction and of the minor
`fraction containing glyco-a-lactalbumin. For comparative pur-
`poses, an o-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 o-lactalbumin was per-
`formed essentially as described by Davies (1974) with some
`modifications. The a-lactalbumin-enriched wheyprotein frac-
`tion (1 g dissolved in 3 mL of elution buffer, 0.01 M
`imidazole-HCI/0.2 M NaCl/0.02% NaN3, pH 6.65) was carefully
`loaded onto the top of a 140 = 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“!.
`Absorbance was continuously measured at 280 nm using a
`Uvicord absorptiometer, type 8303A (LKB, Bromma, Sweden).
`Peakfractions werecollected, dialyzed againstdistilled 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 o-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 o-Lactalbumin and
`Acetylation of Released Oligosaccharides. Deglycosyla-
`tion of glyco-a-lactalbumin wascarried out with PNGase F as
`described by Rademaker and Thomas-Oates (1996). The gly-
`coprotein (0.33 mg dissolved in 200 «L of 50 mM ammonium
`bicarbonate buffer, pH 8.4, containing 1 drop of toluene) was
`incubated with 5 wL (1 unit) of PNGase F solution at 37 °C
`for 16 h. The resulting mixture of deglycosylated o-lactalbumin
`and released oligosaccharides was freeze-dried, redissolved in
`500 uwL 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 mixtureof trifluoroacetic anhydride andacetic
`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-a-lactalbumin was
`obtained from a second eluate fraction (20-40% 1-propanol
`in 5% acetic acid) during the Sep-Pak Plus C18 separation.
`MassSpectrometry.For the analysis of the a-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™ through the
`electrospray interface. Nitrogen was used as a nebulizing and
`drying gas. The capillary was held at 3.9 kV; the cone voltage
`was40 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. Masscalibration was performed
`by multiple-ion monitoring of a Nal/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 a-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 (6-lactoglobulin), IV (mainly glyco-a-lact-
`albumin), and V (nonglyco-a-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, y-globulins, and serum albumin
`(Davies, 1974). Particularly when focusing on a satisfac-
`
`Page 2
`
`

`

`MS Study of a-Lactalbumin Heterogeneity
`
`J. Agric. Food Chem., Vol. 47, No. 11, 1999
`
`4551
`
`
`
`Absorbance(220nm)
`
`0
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`Elution time (min)
`Figure 2. Reversed-phase HPLC patterns of (a) peak componentIV of Figure 1, (b) peak component V of Figure 1, and (c) a
`sample of a-lactalbumin prepared by DEAE-Sepharose chromatography on a technical scale. Peak material was collected and
`used for mass spectrometric analysis.
`
`tory separation between glyco-a-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 S; 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 a-lactalbumin preparation
`produced from cheese whey by DEAE-Sepharose chro-
`matography (trace c)
`(Schmidt and van Markwijk,
`1993). Glyco-a-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 a-lactalbumin derivative (see next sec-
`tion), we may conclude that, on the basis of the above
`yields of fractions ITV and V, ca. 10% (m/m) of a-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 percentageonly slightly exceeds the 7%
`content reported by Baumy and Fauquant (1989), who
`used Sephadex G-50 chromatography and started from
`an industrial a-lactalbumin preparation. The minor
`componenteluting just before the main onein trace c
`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 a-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 o-Lactalbumin Compo-
`nents and Released Oligosaccharides. Massspectra
`and deconvoluted spectra of the main RP-HPLC com-
`ponents representing glyco- and nonglyco-a-lactalbumin
`(see Figure 2) are depicted in Figures 3—5. It was found
`that nonglyco-a-lactalbumin had a mass of 14 178.8 Da
`(Figure 3), which was in agreement with the theoretical
`value of 14 178 Da (Wang etal., 1989). The minor peak
`following the main one in the RP-HPLC pattern of
`Figure 2, trace a, cannotbe ascribed to a natural glyco-
`a-lactalbumin form, as the corresponding mass(14 503
`Da) wastoo low to accountfor any oligosaccharide chain
`attached to a-lactalbumin (result not shown). We have
`experimental evidence now that this componentresults
`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 $-lactoglobulin (Burret al.,
`1996; Léonil et al., 1997). They can be considered as
`early Maillard products. A similar product, isolated from
`humanblood serum, was found to be a reaction product
`of serum albumin and blood glucose (Bunk, 1997).
`The mass spectrum of glyco-a-lactalbumin (Figure 4)
`wasfairly 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-a-lactalbumin (Barman, 1970; Hindle and
`Wheelock, 1971) and the known core structure of
`N-linked carbohydrates, we were able to match the
`massesof the various components shownin Figure 5 to
`those of 14 glycoforms of a-lactalbumin (Table 1).
`
`Page 3
`
`

`

`4552 J. Agric. Food Chem,, Vol. 47, No. 11, 1999
`
`SsoO
`
`=
`25
`Cc
`®£
`£
`
`1005
`
`J
`
`0-
`
`A10
`1418.8
`
`Ail
`1290.1
`
`|
`
`!
`
`,
`|
`1300
`
`A12
`1182.6
`
`
`
`!
`|
`
`‘
`
`1200
`
`A13
`1091.6
`|
`|
`1100
`
`LL
`1000
`
`1422.6
`
`1400
`
`1500
`
`Ag
`
`Slangen and Visser
`
`14178.8
`
`100.
`
`%
`
`AB
`1773.4
`
`0.
`14000
`
`1778.3
`
`14250
`
`14500
`
`14750
`
`mass
`15000
`
`1580.7
`
`
`
`
`i
`
` mz
`
`1700
`1800
`1900
`2000
`2100
`2200
`
`Figure 3. Electrospray mass spectrum of purified nonglyco-a-lactalbumin (main peak in Figure 2, trace b). Charge numbers of
`the molecular-ion clusters are also indicated, with a maximum massintensity at nine charges (A9). The inset shows the deconvoluted
`spectrum.
`
`100-
`
`B10
`1629.1
`
`BO
`
`Ag
`t7an1810.0
`\
`ce
`1814.4
`
`A10|4633.0
`1604.0|/P°*
`
`BS
`11 1589.4||
`
`A&8
`2036.0
`At
`1481.0
`2004.6
`1827.2
`c8
`
`1576.4 1600
`
`
`
`
`
`
`
`
`
`
`
`|
`
`i
`
`|
`
`1765.8
`\
`
`1822.7
`/
`
`| a
`
`2043.1
`|,
`2050.9
`j
`2086.6
`
`1986.5
`
`|
`|
`‘a
`ea
`Ha ae
`ee Lh
`, etd "
`2000
`2050
`2100
`1950
`2150
`
`mz
`
`2200
`
`1856.8
`1860.6
`1863.8
`
`he
`F
`un
`1900
`
`1650 1700
`
`1750
`
`|
`|
`|!
`
`|
`
`|
`
`i |
`
`{
`
`1585.4]
`
`|} G41
`
`1484.7
`co
`
`14583
`
`
`1445.1
`‘
`
`|
`
`1400
`
`i
`1450
`
`H
`
`1491.3
`
`1518.1
`|
`|
`|
`i
`
`1500
`
`|
`J
`
`:
`1550
`
`1600
`
`= g
`
`=~
`
`zsdCc
`2
`
`At2
`B12
`1338843675
`|
`|
`f ;
`ie
`1350
`
`ie
`
`0
`1300
`
`Figure 4. Electrospray mass spectrum of purified glyco-a-lactalbumin (main peak in Figure 2, trace a). Ax, Bx, and Cx denote
`components of the three main envelopes of the spectrum.
`
`Ourresults indicate that 8 of the 14 proposed glyco-
`forms concern sialylated oligossacharide chains. This
`distinction between sialylated and nonsialylated oli-
`
`gosaccharides would accountfor the presenceof the two
`PAS-positive bands in the PAGE-pattern of glyco-a-
`lactalbumin (Figure 1) (Hopper and McKenzie, 1973).
`
`Page 4
`
`

`

`MS Study of a-Lactalbumin Heterogeneity
`
`J. Agric. Food Chem., Vol. 47, No. 11, 1999 4553
`
`
`
`
`
`
`
`
`
`
`
`
`
`0 TTTTyrrrraa Tr T T TTT TT wT TT T TT TT yy T rrr yr ry T T a a T
`
`
`
`
`15600
`15700
`15800
`15900
`16000
`16100
`16200
`16300
`16400
`16500
`16600
`16706
`16800
`
`Figure 5. Deconvoluted mass spectrum of purified glyco-a-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
`a-Lactalbumin#
`
`structure published so far (Tilley et al., 1991) and
`reported to represent the major glycoform
`
`Mass(Da)
`
`GalNAc- GlcNAc-Man
`
`GalNAc-GleNAc-Man
`
`Fuc
`
`Man-GicNAc-GleNAc-Asn,,,
`
`corresponds with that of component 5 in Figure 5 and
`Table 1; this component wasalso 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; Girardetet al., 1995), closely similar
`structures, being either fucosylated or nonfucosylated,
`sialylated or nonsialylated, and having in a numberof
`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-a-
`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;
`Neelemanet al., 1994).
`Molecular masses of glyco-a-lactalbumin reported in
`the literature were calculated from amino acid and
`
`Page 5
`
`monosaccharide
`composition
`based on
`measured mass
`
`LoLAWLOWAWHOW
`
`RBPRORRFREROORKRHO
`
`
`
`PERFORPFPREEFFROOOCOO
`
`NOonrnnobnunuleu
`
`))))))
`
`.
`calcd residue
`massof glycan HexN Fuc Hex NeuAc
` 15842.62 1664.54 (2691.45)
`0
`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
`
`calcd
`mass
`
`measured
`mass
`
`25
`
`
`
`SOONODULWNHE
`
`10
`11
`12
`13.
`14
`
`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
`
`4 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 a-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-a-
`lactalbumin forms in a quantitative manner. On the
`other hand, the mass of the only glyco-a-lactalbumin
`
`100-
`
`
`
`Intensity(%)
`
`
`
`16030
`
`16280
`
`10
`16320
`
`
`
`
`

`

`Slangen and Visser
`
`S=
`&
`3296
`
`Ss
`a
`20
`—
`n
`3254
`
`Ss
`7
`an
`xe
`oS
`3338
`
`~~
`
`Sae
`
`3137
`
`3393
`
`os
`aS
`“AN
`nN
`ZS &¢6
`3571." =
`3529 9°"
`3641 5
`\ 3683
`3487
`
`ce
`S
`4a
`a}
`S
`qn
`=
`=
`~
`on
`—_
`Q
`Oo
`N
`3212
`a
`x
`2606|2690 a 3024
`
`
`a=
`a =
`a
`~
`a
`=
`2
`~
`3411
`3453
`ro
`ow
`o
`Nd
`o
`(
`3871 ©
`2794
`o
`3912
`2564)
`2982
`
`4554 J. Agric. Food Chem,, Vol. 47, No. 11, 1999
`
`100:
`
`-—
`=~
`Xo
`~~
`=
`3066
`
`a
`2
`S
`
`o”<2
`
`2878
`
`>
`nn,
`=
`a)
`a
`2836
`
`o
`*
`wt
`&
`o
`2920
`
`=
`=B
`Ss
`2£
`i=
`—
`
`a
`NX
`=
`oS
`-
`2648
`
`2507
`
`0.
`
`2500
`
`2600
`
`2700
`
`2800
`
`2900
`
`3000
`
`3100
`
`3200
`
`3300
`
`3400
`
`3500
`
`3600
`
`3700
`
`3800
`
`3900
`
`Mass(Da)
`
`Figure 6. Deconvoluted mass spectrum of enzymatically released and peracetylated oligosaccharides originating from glyco-a-
`lactalbumin. Number of acetyl groups missing as a result of incomplete derivatization are indicated by x in x(y), 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.
`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 glycoformspostulated in Table 1 were supported
`by mass spectrometric analysis of the oligosaccharide
`fraction obtained after deglycosylation of glyco-a-lact-
`albumin with PNGase F and subsequentperacetylation.
`No distinction could be made betweenseveral acetylated
`oligosaccharides of similar mass. Acetylation wascar-
`ried out in order to allow the subsequent extraction of
`derivatized oligosaccharides into the organic phase,
`which offers a moreeffective 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-a-lactalbumin was found to
`be 14 178.5 Da (calculated: 14 179 Da for a-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 Figure5.
`The fact that complete deglycosylation could be achieved
`with nondenatured glyco-a-lactalbumin indicates that
`the protein—glycan linkageis 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) a-lactalbumin molecule.
`For comparison, we have also analyzed the minor
`peak preceding the major one in trace c of Figure 2. It
`represented the glycoform of an a-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-a-lactalbumin
`preparation appeared to contain only part of the
`glycoforms shown in Table 1 (mainly components1, 6,
`7, 9, and 10; result not shown). Therefore, it is likely
`that the numberandratio of glyco-a-lactalbumin forms
`found depend on the material used and, more particu-
`larly, on the modeof its isolation and purification.
`In summary,
`it may be concluded that our mass
`spectrometric analysis of the nonglycosylated fraction
`of bovine a-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-
`a-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
`concernsclosely similar structures, either fucosylated
`
`Page 6
`
`

`

`MS Study of a-Lactalbumin Heterogeneity
`
`J. Agric. Food Chem., Vol. 47, No. 11, 1999 4555
`
`or nonfucosylated and sialylated or nonsialylated,
`whereas in quite a numberof cases Gal is replaced by
`GalNAc.
`While data such as overall carbohydrate composition
`and single, N-linked glycosylation site were already
`knownfrom theliterature, our study demonstrates that
`mass spectrometry is a powerful tool to gain further
`insight
`into the structural composition of glyco-a-
`lactalbumin. In ourcase 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-MSanalysis 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-a-lactalbumin
`fraction still remains unclear. It has been suggested
`(Barman, 1970) that in the Golgi region of the mammary
`gland cells a-lactalbumin is wholly secreted as a glyco-
`protein and subsequently degraded by milk glycoside
`hydrolases. If this would be the case, then thebiological
`significance of the glycan moiety in o-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-
`N*(N-acetyl-£-glucosaminyl)asparagine amidase F; RP-
`HPLC, reversed-phase high-performance chromatogra-
`phy.
`
`ACKNOWLEDGMENT
`
`Wethank Peter van Rooijen for the isolation of glyco-
`a-lactalbumin and Janneke Kromkampfor providing us
`with an a-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-a-lactalbumin components
`1-14 based on the results presented in Figure 5 and Table 1
`of this paper.
`
`LITERATURE CITED
`
`Armstrong, J. McD.; Hopper, K. E.; McKenzie, H. A.; Murphy,
`W. H. On the column chromatography of bovine whey
`proteins. Biochim. Biophys. Acta 1970, 214, 419-426.
`
`Improved method for the
`Aschaffenburg, R.; Drewry, J.
`preparation of crystalline 6-lactoglobulin and a-lactalbumin
`from cow’s milk. Biochem. J. 1957, 65, 273-277.
`Barman, T. E. Purification and properties of bovine milk glyco-
`a-lactalbumin. Biochim. Biophys. Acta 1970, 214, 242-244.
`Baumy, J. J.; Fauquant, J. Mise en évidence d’o-lactalbumine
`glycosylée dans une préparation industrielle d’a-lactalbu-
`mine bovine. Lait 1989, 69, 315-322.
`Brew, K.; Grobler, J. A. a-Lactalbumin. In Advanced Dairy
`Chemistry; Fox, P. F., Ed.; Elsevier Applied Science: Lon-
`don, 1992; Vol. 1, pp 191-229.
`Brodbeck, U.; Ebner, K. E. Resolution of a soluble lactose
`synthetase into two protein components and solubilization
`of microsomallactose synthetase. /. Biol. Chem. 1966, 241,
`762-764.
`
`Bunk, D. M. Characterization of the glycation of albumin in
`freeze-dried and frozen human serum. Anal. Chem. 1997,
`69, 2457-2463.
`Burr, R.; Moore, C. H.; Hill, J. P. Evidence of multiple
`glycosylation of bovine f-lactoglobulin by electrospray ion-
`ization mass spectrometry. Milchwissenschaft 1996, 51,
`488-492.
`Coddeville, B.; Strecker, G.; Wieruszeski, J. M.; Vliegenthart,
`J. F. G.; van Halbeek, H.; Peter-Katalinic, J.; Egge, H.; Spik,
`G. Heterogeneity of bovine lactotransferrin glycans. Char-
`acterization of o-D-Gal p-(1—3)-8-D-Gal- and a-NeuAc—
`(2—-6)-8-D-Gal pNAc—(1—4)-8-D-GlcNAc-substituted N-
`linked glycans. Carbohydr. Res. 1992, 236, 145-164.
`Davies, D. T. The quantitative partition of the albumin fraction
`of milk serum proteins by gel chromatography. 7. Dairy Res.
`1974, 41, 217-228.
`Ebner, K. E.; Denton, W. L.; Brodbeck, U. The substitution of
`a-lactalbumin for the B protein of lactose synthetase.
`Biochem. Biophys. Res. Commun. 1966, 24, 232-236.
`Girardet, J.-M.; Linden, G. PP3 componentof bovine milk: a
`phosphorylated whey glycoprotein. J. Dairy Res. 1996, 63,
`333-350.
`Girardet, J.-M.; Coddeville, B.; Plancke, Y.; Strecker, G.;
`Campagna, S.; Spik, G.; Linden, G. Structure of glycopep-
`tides isolated from bovine milk component PP3. Fur. J.
`Biochem. 1995, 234, 939-946.
`Green,B. N.; Hutton, T.; Vinogradov, S. N. Analysis of complex
`protein and glycoprotein mixtures by electrospray ionization
`mass spectrometry with maximum entropy processing. In
`Methods in Molecular Biology (Protein and Peptide Analysis
`by Mass Spectrometry), Chapman J. R., Ed.; Humana Press
`Inc.: Totowa, NJ, 1996; Vol. 61, pp 279-294.
`Harvey, D. J. Identification of cleaved oligosaccharides by
`matrix-assisted laser desorption/ionisation. In Methods in
`Molecular Biology (Protein and Peptide Analysis by Mass
`Spectrometry), Chapman J. R., Ed.; Humana Press Inc.:
`Totowa, NJ, 1996; Vol. 61, pp 243-253.
`Hill, R. L.; Brew, K. Lactose synthetase. Adv. Enzymol. Relat.
`Areas Mol. Biol. 1975, 43, 411-490.
`Hindle, E. J.; Wheelock, J. V. Carbohydrates of bovine a-
`lactalbumin preparations. Chimia 1971, 25, 188-190.
`Hopper, K. E.; McKenzie, H. A. Minor components of bovine
`a-lactalbumin A and B. Biochim. Biophys. Acta 1973, 295,
`352-363.
`Kapitany, R. A.; Zebrowski, E. J. A high-resolution PAS stain
`for polyacrylamide gel electrophoresis. Anal. Biochem. 1973,
`56, 361-369.
`Léonil, J.; Mollé, D.; Fauquant, J.; Maubois, J. L.; Pearce, R.
`J.; Bouhallab, S. Characterization by ionization mass spec-
`trometry of lactosyl f-lactoglobulin conjugates formed dur-
`ing heat treatment of milk and whey andidentification of
`one lactose-binding site. J. Dairy Sci. 1997, 80, 2270—2281.
`Montreuil, J. Primary structure of glycoprotein glycans. Basis
`for the molecular biology of glycoproteins. In Advances in
`Carbohydrate Chemistry and Biochemistry, Tipson, R. S.,
`Horton, D., Eds.; Academic Press: New York, 1980; Vol. 37,
`pp 157-223.
`Neeleman, A. P.; van der Knaap, W.P. W.; van den Eijnden,
`D. H. Identification and characterization of a UDP—GalNAc:
`GlcNAcf-Rf1—4-N-acetylgalactosaminyltransferase from
`
`Page 7
`
`

`

`4556 J. Agric. Food Chem,, Vol. 47, No. 11, 1999
`
`cercariae of the schistosome Trichobilharzia ocellata. Ca-
`talysis of a key step in the synthesis of N,N -diacetyllac-
`tosediamino (lacdiNAc)-type glycans. Glycobiology 1994, 4,
`641-651.
`
`Pike, A. C. W.; Brew, K.; Acharya, K. R. Crystal structures of
`guinea-pig, goat and bovine a-lactalbumin highlight the
`enhanced conformational flexibility of regions that are
`significant for its action in lactose synthase. Structure 1996,
`4, 691-703 (Brookhaven PDB, code 1 HFZ).
`Rademaker,G. J.; Thomas-Oates, J. Analys