Large membrane-associated oligosaccharides linked to ceramide, named megaloglycolipids or macroglycolipids, and carrying various blood group activities were first proposed by Gardas and Koscielak (Gardas and Koscielak, 1973). Preparations of human erythrocytes contained 90% carbohydrate, 7% amino acids, and about 2% sphingosine. The glycoconjugates were concluded to be unusually complex glycoproteins or glycolipids containing 30-50 saccharide residues per ceramide. The amino acid contents could be reduced to about 0.3% by alkali treatment (Gardas and Koscielak, 1974) which indicated the presence of glycolipids. Koscielak et al. (Koscielak et al., 1976) introduced the name polyglycosylceramides (PGCs) for these polar glycosphingolipids. Structural studies of a component isolated from blood group O erythrocytes provided evidence for a 22-sugar glycosphingolipid composed of eight N-acetyllactosamines in a straight chain with two branches of N-acetyllactosamine carrying blood group O determinants (Gardas, 1976a). Partial degradation of this preparation with fucosidase gave a product highly active with anti-i antibodies (Gardas, 1976b). A glycosphingolipid isolated from blood group A erythrocytes was proposed to contain 23 sugar residues and ending with blood group O and blood group A determinants (Gardas, 1978). Subsequent studies provided evidence for a high microheterogeneity of such polar preparations of human erythrocytes (Dejter-Juszynski et al., 1978; Koscielak et al., 1979; Fukuda and Hakomori, 1982; Zdebska et al., 1983; Hanfland et al., 1984).
Corresponding rabbit erythrocyte preparations revealed a more distinct, stepwise glycosylation (Hanfland et al., 1981). Ceramide-linked saccharides containing 10 (Hanfland et al., 1981), 15 (Dabrowski et al., 1984; Egge et al., 1985), 20 and 25 (Hanfland et al., 1988), 30, 35, and 40 (Kordowicz et al. 1986) monosaccharides could be differentiated. A complete structure of the 40-sugar glycolipid was determined with the aid of 600 MHz two-dimensional 1H NMR spectroscopy (Dabrowski et al., 1988). This glycolipid was an octa-antennary, regularly branched N-acetyllactosamine sequence ending with Gal[alpha]3 residues. Analogous structures with up to 20 sugars terminating with NeuAc[alpha]3 have been characterized from human placenta (Levery et al., 1989). More recently linear, multifucosylated poly-N-acetyllactosamine glycolipids with up to 16 sugars containing NeuAc[alpha]3 have been isolated from human leukocytes (Müthing et al., 1996; Stroud et al., 1996a,b).
Our own present interest in PGCs was aroused by the finding that the human gastric pathogen Helicobacter pylori was binding with high affinity to PGCs of human erythrocytes (Miller-Podraza et al., 1996, 1997b). The binding vanished upon treatment with neuraminidase, weak acid or mild periodate oxidation indicating a dependence of binding on sialic acid. Apparently, the binding was specific for human PGCs since preparations from animal sources, although containing sialic acid, were not recognized by the bacterium (Miller-Podraza et al., 1997a,c). A large number of other sialic acid-containing glycolipids and glycoproteins of human and animal origins were not recognized. At present, the only object for structural studies of the binding epitope is therefore human PGCs. To plan laborious synthesis and modeling studies, an average structure based on simply composition analysis is not enough. Binding and modification studies (Miller-Podraza et al., 1996, 1997a,b) must be related to a precisely defined sequence. Therefore subfractionation of complex mixtures of PGCs or enzymatic synthesis of defined carbohydrate structures will be necessary for final determination of the binding epitope.
Matrix-assisted laser desorption/ionization and electrospray are ionization techniques available for analysis of larger glycoconjugates. Ii et al. have used electrospray mass spectrometry (Ii et al., 1993) for the analysis of a synthetic 25-sugar saccharide of the type present in rabbit erythrocytes (Hanfland et al., 1988). The measured average molecular masses were 4441 Da and 9416 Da for the nonderivatized and the acetylated saccharide, respectively. However, this interesting approach is not applicable in our case due to the complexity of the mixtures.
The present paper reports that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a good method for detection of molecular species of PGCs in complex mixtures. The sample used was PGCs of human blood group O erythrocytes, corresponding to the preparation initially described (Gardas and Koscielak, 1973, 1974). However, in our case a rationalized and simplified preparative approach was used based on peracetylation (Miller-Podraza et al., 1993). This resulted in a complex PGC fraction suitable for further studies. The MALDI mass spectra of both neutral and sialylated glycosphingolipids revealed complex series with increasing number of N-acetyllactosamines and degree of fucosylation. The number of sugar residues ranged from about 10 up to 41, with a ceramide composed of mainly sphingosine in combination with 24:0 or 24:1 nonhydroxy fatty acid. The data supported earlier proposals of a successively extended and branched poly-N-acetyllactosamine core structure with terminal substitutions of Fuc and NeuAc in different combinations (Koscielak et al., 1976).
The present work was performed on PGC material isolated from human blood group O erythrocytes. Four subfractions were analyzed in parallel: fraction 1 and 2 containing polyglycosylceramides and fraction A and B containing oligosaccharides. The PGC fractions were obtained by separation of acetylated derivatives on silica gel columns. Oligosaccharides were enzymatically released by ceramide glycanase from a mixture of PGCs. The oligosaccharides were separated by high-pH anion-exchange chromatography (HPAEC) and two of the eluted fractions were further analyzed. The chemical composition of the four fractions are summarized in Table
Figure 1. Thin-layer chromatograms (silica gel 60 TLC plates, Merck, Germany) of fraction 2 of polyglycosylceramides (lane 1) and brain gangliosides (lane 2) detected with anisaldehyde (left) or by autoradiography after overlay with radiolabeled Helicobacter pylori cells (right). Developing solvent was propanol/0.25% KCl in water/methanol/chloroform, 7:5:1:0.5 (by vol.).
Table I.
Fraction
Hex/SFb
SA/SF
Hex/SA
Polyglycosylceramides
Fraction 1
11.6
0.5
23.2
Fraction 2
13.3
1.6
8.3
Oligosaccharides
Fraction A
73.5
Fraction B
12.7
Glycosphingolipids from human sources are composed of a ceramide part and relatively few different monosaccharides with unique incremental masses (e.g., fucose, 146.1 Da; hexose, 162.1 Da; N-acetylhexosamine, 203.2 Da; N-acetylneuraminic acid, 291.3 Da; average masses). The molecular weights provided from the MALDI mass spectra recorded both in the linear and reflectron mode with an accuracy of [le]0.08% and [le]0.02%, respectively, allowed determination of the saccharide compositions. Higher masses were detected in the linear mode with a more symmetrical mass distribution envelope. These mass spectra probably reflected the true contents of the fractions better than the data obtained in reflectron mode. The detected ions in the reflectron mode were shifted towards lower mass ions, but the resolution was higher and the mass measurements were more accurate. Neutral components were detected as [M+Na]+ in the positive ion mode, and sialylated species were detected as [M-H]- in the negative ion mode.
Fraction 1 with less sialylated PGCs
The MALDI-TOF mass spectra in positive ion linear and reflectron mode of fraction 1 are shown in Figure
Figure 2. MALDI-TOF mass spectrum of fraction 1 in (A) positive ion linear mode and (B) positive ion reflectron mode. The annotations are made as HexNAc.Fuc (Tables II and III). The * indicates the less abundant d18:1-22:0 ceramide. Fraction 2 with more sialylated PGCs
MALDI-TOF mass spectra of the more sialylated second fraction were only observed in negative ion linear mode (Figure
Table II.
Table III.
Structuree
In Fig.f
Formula
CMb [M+Na]+
OM (linear) [M+Na]+
[Delta] (linear)
OM (reflectr.) [M+Na]+
[Delta] (reflectr.)
(6,4,1)Cer
4.1
C116H205(203)N5O57
2603.89
2605.3
+1.4
2604.85
+0.96
(6,4,2)Cer
4.2
C122H215(213)N5O61
2750.03
2749.8
-0.2
2750.65
+0.62
(7,5,0)Cer
5.0
C124H218(216)N6O63
2823.08
2824.9
+1.8
2823.62
+0.54
(7,5,1)Cer
5.1
C130H228(226)N6O67
2969.22
2969.6
+0.4
2969.90
+0.68
(7,5,2)Cer
5.2
C136H238(236)N6O71
3115.37
3115.5
+0.1
3115.67
+0.30
(8,6,0)Cer
6.0
C138H241(239)N7O73
3188.42
3189.4
+1.0
3189.14
+0.72
(8,6,1)Cer
6.1
C144H251(249)N7O77
3334.56
3335.1
+0.6
3335.18
+0.62
(8,6,2)Cer
6.2
C150H261(259)N7O81
3480.70
3481.0
+0.3
3481.15
+0.45
(8,6,3)Cer
6.3
C156H271(269)N7O85
3626.85
3626.5
-0.4
3627.24
+0.39
(9,7,0)Cer
7.0
C152H264(262)N8O83
3553.76
3553.3
-0.5
3554.11
+0.35
(9,7,1)Cer
7.1
C158H274(272)N8O87
3699.90
3700.8
+0.9
3700.67
+0.77
(9,7,2)Cer
7.2
C164H284(282)N8O91
3846.04
3846.7
+0.7
3846.36
+0.32
(9,7,3)Cer
7.3
C170H294(292)N8O95
3992.19
3992.6
+0.4
3992.74
+0.55
(9,7,4)Cer
7.4
C176H304(302)N8O99
4138.33
4137.9
-0.4
4138.85
+0.52
(10,8,0)Cer
8.0
C166H287(285)N9093
3919.09
-
-
3919.87
+0.78
(10,8,1)Cer
8.1
C172H297(295)N9O97
4065.24
4065.2
0.0
4065.91
+0.67
(10,8,2)Cer
8.2
C178H307(305)N9O101
4211.38
4211.6
+0.2
4211.83
+0.45
(10,8,3)Cer
8.3
C184H317(315)N9O105
4357.52
4357.4
-0.1
4358.12
+0.60
(10,8,4)Cer
8.4
C190H327(325)N9O109
4503.67
4503.1
-0.6
4504.17
+0.50
(11,9,1)Cer
9.1
C186H320(318)N10O107
4430.57
4431.7
+1.1
4430.49
-0.08
(11,9,2)Cer
9.2
C192H330(328)N10O111
4576.72
4576.3
-0.4
4576.98
+0.26
(11,9,3)Cer
9.3
C198H340(338)N10O115
4722.86
4722.7
-0.2
4723.62
+0.76
(11,9,4)Cer
9.4
C204H350(348)N10O119
4869.00
4868.4
-0.6
4869.42
+0.42
(11,9,5)Cer
9.5
C210H360(358)N10O123
5015.15c
5013.9
-1.3
5015.20
+0.05
(12,10,0)Cer
10.0
C194H333(331)N11O113
4649.77
-
-
4649.66
-0.11
(12,10,1)Cer
10.1
C200H343(341)N11O117
4795.91
-
-
4796.63
+0.72
(12,10,2)Cer
10.2
C206H353(351)N11O121
4942.05
4941.7
-0.4
4942.22
+0.17
(12,10,3)Cer
10.3
C212H363(361)N11O125
5088.20
5086.5
-1.7
5088.57
+0.37
(12,10,4)Cer
10.4
C218H373(371)N11O129
5234.34
5233.6
-0.7
5235.32
+0.98
(12,10,5)Cer
10.5
C224H383(381)N11O133
5380.48c
5379.2
-1.3
5380.83
+0.35
(13,11,3)Cer
11.3
C226H386(384)N12O135
5453.53
5452.3
-1.2
-
-
(13,11,4)Cer
11.4
C232H396(394)N12O139
5599.68
5598.8
-0.9
5599.31
-0.37
(13,11,5)Cer
11.5
C238H406(404)N12O143
5745.82c
5744.5
-1.3
5745.20
-0.62
(13,11,6)Cer
11.6
C244H416(414)N12O147
5891.96c
5889.9
-2.1
5892.00
+0.04
(14,12,4)Cer
12.4
C246H419(417)N13O149
5965.02
5961.1
-3.9
5964.81
-0.21
(14,12,5)Cer
12.5
C252H429(427)N13O153
6111.16c
6109.0
-2.2
-
-
(14,12,6)Cer
12.6
C258H439(437)N13O157
6257.30c
6257.5
+0.2
6257.50
+0.20
(15,13,4)Cer
13.4
C260H442(440)N14O159
6330.35
6331.7
+1.4
-
-
(15,13,5)Cer
13.5
C266H452(450)N14O163
6476.50c
6474.1
-2.4
6476.62
+0.12
(15,13,6)Cer
13.6
C272H462(460)N14O167
6622.64c
6620.1
-2.5
6623.43
+0.79
(16,14,6)Cer
14.6
C286H485(483)N15O177
6987.98c
6987.1
-0.9
-
-
(16,14,7)Cer
14.7
C292H495(493)N15O181
7134.13c
7137.1
+3.0
-
-
(17,15,6)Cer
15.6
C300H508(506)N16O187
7353.31c
7352.9
-0.4
-
-
n = 40d
[sigma] = 1.3d
n = 37d
[sigma] = 0.36d
Accuracy:
0.02-0.05%
0.006-0.014%
Structuree
Formula
CMb [M+Na]+
OM (linear) [M+Na]+
[Delta] (linear)
OM (reflectron) [M+Na]+
[Delta] (reflectron)
(6,4,1)Cer
C114H201N5O57
2576.84
2577.8
+1.0
-
-
(7,5,2)Cer
C134H234N6O71
3088.32
3089.5
+1.2
-
-
(8,6,0)Cer
C136H237N7O73
3161.37
3161.8
+0.4
-
-
(8,6,1)Cer
C142H247N7O77
3307.51
-
-
-
-
(8,6,2)Cer
C148H257N7O81
3453.66
3454.5
+0.8
3453.67
+0.01
(8,6,3)Cer
C154H267N7O85
3599.80
3599.4
-0.4
3600.89
+1.09
(9,7,1)Cer
C156H270N8O87
3672.85
3671.3
-1.6
3672.65
-0.20
(9,7,2)Cer
C162H280N8O91
3818.99
3820.6
+1.6
3818.51
-0.48
(9,7,3)Cer
C168H290N8O95
3965.14
3966.0
+0.9
3965.43
+0.29
(10,8,2)Cer
C176H303N9O101
4184.33
4183.5
-0.8
-
-
(10,8,3)Cer
C182H313N9O105
4330.47
4330.2
-0.3
4330.75
+0.28
(10,8,4)Cer
C188H323N9O109
4476.62
4477.4
+0.8
4476.92
+0.30
(11,9,3)Cer
C196H336N10O115
4695.81
4694.0
-1.8
-
-
(11,9,4)Cer
C202H346N10O119
4841.96
4841.6
-0.4
4842.24
+0.28
(12,10,3)Cer
C210H359N11O125
5061.15
-
-
5062.00
+0.85
(12,10,4)Cer
C216H369N11O129
5207.29
5204.2
-3.1
5207.19
-0.10
(12,10,5)Cer
C222H379N11O133
5353.44c
-
-
5353.49
+0.05
(13,11,4)Cer
C230H392N12O139
5572.63
-
-
5572.61
-0.02
(13,11,5)Cer
C236H402N12O147
5718.77c
-
-
5718.05
-0.72
n = 14d
[sigma] = 1.3d
n = 13d
[sigma] = 0.49d
Accuracy:
0.03-0.05%
0.009-0.014%
Figure 3. MALDI-TOF mass spectrum of fraction 2 in the negative ion linear mode showing the monosialylated species annotated as HexNAc.Fuc (Table IV and V). The less abundant ceramide d18:1-22:0 is indicated by *.
Table IV.
Structuree | In Fig.f | Formula | CMb[M-H]- | OM [M-H]- | [Delta] |
(7,5,0,1)Cer | 5.0 | C135H235(233)N7O71 | 3090.34 | 3091.7 | +1.4 |
(8,6,0,1)Cer | 6.0 | C149H258(256)N8O81 | 3455.67 | 3457.4 | +1.7 |
(8,6,1,1)Cer | 6.1 | C155H268(266)N8O85 | 3601.82 | 3602.7 | +0.9 |
(9,7,0,1)Cer | 7.0 | C163H281(279)N9O91 | 3821.01 | 3822.7 | +1.7 |
(9,7,1,1)Cer | 7.1 | C169H291(289)N9O95 | 3967.16 | 3968.6 | +1.4 |
(9,7,2,1)Cer | 7.2 | C175H301(299)N9O99 | 4113.30c | 4112.9 | -0.4 |
(9,7,3,1)Cer | 7.3 | C181H311(309)N9O103 | 4259.44c | 4258.9 | -0.5 |
(10,8,0,1)Cer | 8.0 | C177H304(302)N10O101 | 4186.35 | 4188.6 | +2.3 |
(10,8,1,1)Cer | 8.1 | C183H314(312)N10O105 | 4332.49 | 4333.9 | +1.4 |
(10,8,2,1)Cer | 8.2 | C189H324(322)N10O109 | 4478.64c | 4478.6 | 0.0 |
(10,8,3,1)Cer | 8.3 | C195H334(332)N10O113 | 4624.79c | 4624.5 | -0.3 |
(11,9,0,1)Cer | 9.0 | C191H327(325)N11O111 | 4551.69 | 4553.7 | +2.0 |
(11,9,1,1)Cer | 9.1 | C197H337(335)N11O115 | 4697.83 | 4699.5 | +1.7 |
(11,9,2,1)Cer | 9.2 | C203H347(345)N11O119 | 4843.97c | 4844.8 | +0.8 |
(11,9,3,1)Cer | 9.3 | C209H357(355)N11O123 | 4990.12c | 4990.9 | +0.8 |
(11,9,4,1)Cer | 9.4 | C215H367(365)N11O127 | 5136.26c | 5135.3 | -1.0 |
(12,10,0,1)Cer | 10.0 | C205H350(348)N12O121 | 4917.02 | 4918.2 | +1.2 |
(12,10,1,1)Cer | 10.1 | C211H360(358)N12O125 | 5063.17 | 5064.9 | +1.7 |
(12,10,2,1)Cer | 10.2 | C217H370(368)N12O129 | 5209.31 | 5209.9 | +0.6 |
(12,10,3,1)Cer | 10.3 | C223H380(378)N12O133 | 5355.45 | 5356.7 | +1.3 |
(12,10,4,1)Cer | 10.4 | C229H390(388)N12O137 | 5501.60 | 5501.8 | +0.2 |
(13,11,0,1)Cer | 11.0 | C219H373(371)N13O131 | 5282.36 | 5282.4 | 0.0 |
(13,11,1,1)Cer | 11.1 | C225H383(381)N13O135 | 5428.51 | 5429.7 | +1.2 |
(13,11,2,1)Cer | 11.2 | C231H393(391)N13O139 | 5574.65c | 5575.1 | +0.5 |
(13,11,3,1)Cer | 11.3 | C237H403(401)N13O143 | 5720.79c | 5721.3 | +0.5 |
(13,11,4,1)Cer | 11.4 | C243H413(411)N13O147 | 5866.93c | 5866.7 | -0.2 |
(13,11,5,1)Cer | 11.5 | C249H423(421)N13O151 | 6013.08c | 6012.6 | -0.5 |
(14,12,0,1)Cer | 12.0 | C233H396(394)N14O141 | 5647.70 | 5647.2 | -0.5 |
(14,12,1,1)Cer | 12.1 | C239H406(404)N14O145 | 5793.84 | 5792.9 | -0.9 |
(14,12,2,1)Cer | 12.2 | C245H416(414)N14O149 | 5939.99c | 5940.3 | +0.3 |
(14,12,3,1)Cer | 12.3 | C251H426(424)N14O153 | 6086.13c | 6086.7 | +0.6 |
(14,12,4,1)Cer | 12.4 | C257H436(434)N14O157 | 6232.27c | 6233.0 | +0.7 |
(14,12,5,1)Cer | 12.5 | C263H446(444)N14O161 | 6378.41c | 6378.8 | +0.4 |
(15,13,1,1)Cer | 13.1 | C253H429(427)N15O155 | 6159.18 | 6157.6 | -1.6 |
(15,13,2,1)Cer | 13.2 | C259H439(437)N15O159 | 6305.32c | 6304.8 | -0.5 |
(15,13,3,1)Cer | 13.3 | C265H449(447)N15O163 | 6451.47c | 6451.6 | +0.1 |
(15,13,4,1)Cer | 13.4 | C271H459(457)N15O167 | 6597.61c | 6597.0 | -0.6 |
(15,13,5,1)Cer | 13.5 | C277H469(467)N15O171 | 6743.75c | 6744.3 | +0.6 |
(15,13,6,1)Cer | 13.6 | C283H479(477)N15O175 | 6889.89c | 6889.0 | -0.9 |
(16,14,1,1)Cer | 14.1 | C267H452(450)N16O165 | 6524.52 | 6524.7 | +0.2 |
(16,14,2,1)Cer | 14.2 | C273H462(460)N16O169 | 6670.66c | 6670.1 | -0.6 |
(16,14,3,1)Cer | 14.3 | C279H472(470)N16O173 | 6816.80c | 6815.2 | -1.6 |
(16,14,4,1)Cer | 14.4 | C285H482(480)N16O177 | 6962.95c | 6961.8 | -1.2 |
(16,14,5,1)Cer | 14.5 | C297H492(490)N16O181 | 7109.09c | 7107.9 | -1.2 |
(16,14,6,1)Cer | 14.6 | C297H502(500)N16O185 | 7255.23c | 7255.9 | +0.7 |
(17,15,2,1)Cer | 15.2 | C287H485(483)N17O179 | 7036.00c | 7034.8 | -1.2 |
(17,15,3,1)Cer | 15.3 | C293H495(493)N17O183 | 7182.14c | 7181.6 | -0.5 |
(17,15,4,1)Cer | 15.4 | C299H505(503)N17O187 | 7328.28c | 7328.0 | -0.3 |
(17,15,6,1)Cer | 15.6 | C311H525(523)N17O195 | 7620.57c | 7618.6 | -2.0 |
(18,16,2,1)Cer | 16.2 | C301H508(506)N18O189 | 7401.33c | 7399.4 | -1.9 |
(18,16,3,1)Cer | 16.3 | C307H518(516)N18O193 | 7547.48c | 7545.8 | -1.7 |
(18,16,4,1)Cer | 16.4 | C313H528(526)N18O197 | 7693.62c | 7692.6 | -1.0 |
(18,16,5,1)Cer | 16.5 | C319H538(536)N18O201 | 7839.76c | 7838.0 | -1.8 |
(19,17,3,1)Cer | 17.3 | C321H541(539)N19O203 | 7912.81c | 7911.6 | -1.2 |
(19,17,4,1)Cer | 17.4 | C327H551(549)N19O207 | 8058.96c | 8057.4 | -1.6 |
n = 55d | [sigma] = 1.1d | ||||
Accuracy: | 0.01-0.04% |
Table V.
Structuree | In Fig.f | Formula | CMb [M-H]- | OM [M-H]- | [Delta] |
(8,6,0,1)Cer | 6.0 | C147H254N8O81 | 3428.63 | 3430.5 | +1.9 |
(9,7,0,1)Cer | 7.0 | C161H287N9O95 | 3940.11 | 3942.2 | +2.1 |
(10,8,0,1)Cer | 8.0 | C175H300N10O101 | 4159.31 | 4158.1 | -1.2 |
(10,8,1,1)Cer | 8.1 | C181H310N10O105 | 4305.45 | 4308.4 | +3.0 |
(10,8,2,1)Cer | 8.2 | C187H320N10O109 | 4451.59c | 4451.3 | -0.3 |
(10,8,3,1)Cer | 8.3 | C193H330N10O113 | 4597.73c | 4596.5 | -1.2 |
(11,9,0,1)Cer | 9.0 | C189H323N11O111 | 4524.64 | 4525.5 | +0.9 |
(11,9,1,1)Cer | 9.1 | C195H333N11O115 | 4670.79 | 4672.0 | +1.2 |
(11,9,2,1)Cer | 9.2 | C201H343N11O119 | 4816.93c | 4818.5 | +1.6 |
(11,9,3,1)Cer | 9.3 | C207H353N11O123 | 4963.07c | 4962.4 | -0.7 |
(11,9,4,1)Cer | 9.4 | C213H363N11O127 | 5109.22c | 5107.9 | -1.3 |
(12,10,0,1)Cer | 10.0 | C203H346N12O121 | 4889.98 | 4888.4 | -1.6 |
(12,10,2,1)Cer | 10.2 | C215H366N12O129 | 5182.27c | 5182.9 | +0.6 |
(12,10,3,1)Cer | 10.3 | C221H376N12O133 | 5328.41c | 5328.7 | +0.3 |
(12,10,4,1)Cer | 10.4 | C227H386N12O137 | 5474.55c | 5473.9 | -0.7 |
(13,11,0,1)Cer | 11.0 | C217H369N13O131 | 5255.32 | 5252.7 | -2.6 |
(13,11,2,1)Cer | 11.2 | C229H389N13O139 | 5547.60c | 5546.8 | -0.8 |
(13,11,3,1)Cer | 11.3 | C235H399N13O143 | 5693.75c | 5691.9 | -1.9 |
(13,11,4,1)Cer | 11.4 | C241H409N13O147 | 5839.89c | 5840.9 | +1.0 |
(14,12,3,1)Cer | 12.3 | C249H422N14O153 | 6059.08c | 6057.0 | -2.1 |
(14,12,4,1)Cer | 12.4 | C255H432N14O157 | 6205.23c | 6205.7 | +0.5 |
(15,13,4,1)Cer | 13.4 | C269H455N15O167 | 6570.56c | 6573.0 | +2.4 |
(15,13,6,1)Cer | 13.6 | C281H475N15O175 | 6862.85c | 6861.6 | -1.3 |
(16,14,3,1)Cer | 14.3 | C277H468N16O173 | 6789.76c | 6790.1 | +0.3 |
(16,14,6,1)Cer | 14.6 | C295H498N16O185 | 7228.19c | 7226.3 | -1.9 |
(17,15,1,1)Cer | 15.1 | C279H471N17O175 | 6862.81c | 6861.6 | -1.2 |
(17,15,6,1)Cer | 15.6 | C309H521N17O195 | 7593.53c | 7594.2 | +0.7 |
n = 27d | [sigma] = 1.5d | ||||
Accuracy: | 0.02-0.04% |
Fraction A with free oligosaccharides of low sialic acid content
Fraction A with a low sialic acid content (Table VI) and ions of the neutral species were observed in both positive ion linear and reflectron mode (Figure
Fraction B with free oligosaccharides of higher sialic acid content
Fraction B contained sialic acid corresponding to monosialylated oligosaccharides (Table VII). The [M-H]- ions produced by MALDI were detected in negative ion linear and reflectron mode. The observed series of the general formula Hex(x+2)HexNAc(x)Fuc(y)NeuAc(1)Cer, with x varying from 6 to 16 and y from 0 to 7, were in agreement with those of fraction 2 of intact polyglycosylceramides. Series from (8,6,0,1) up to (18,16,6,1) and up to (17,15,6,1) were observed in linear and reflectron mode, respectively (Table VII and Figure
Structuree | In Fig.f | Formula | CMb [M+Na]+ | OM (linear) [M+Na]+ | [Delta] (linear) | OM (reflectron) [M+Na]+ | [Delta] (reflectron) |
(6,4,0) | 4.0 | C68H114N4O51 | 1826.64 | 1826.4 | -0.2 | 1826.31 | -0.33 |
(6,4,1) | 4.1 | C74H124N4O55 | 1972.78 | 1972.2 | -0.6 | 1972.27 | -0.51 |
(6,4,2) | 4.2 | C80H134N4O59 | 2118.93 | 2117.6 | -1.3 | 2118.96 | +0.03 |
(7,5,0) | 5.0 | C82H137N5O61 | 2191.98 | 2191.2 | -0.8 | 2191.25 | -0.73 |
(7,5,1) | 5.1 | C88H147N5O65 | 2338.12 | 2338.6 | +0.5 | 2337.63 | -0.49 |
(7,5,2) | 5.2 | C94H157N5O69 | 2484.26 | - | - | 2483.79 | -0.47 |
(8,6,0) | 6.0 | C96H160N6O71 | 2557.31 | - | - | 2557.12 | -0.19 |
(8,6,1) | 6.1 | C102H170N6O75 | 2703.46 | 2703.3 | -0.2 | 2703.27 | -0.19 |
(8,6,2) | 6.2 | C108H180N6O79 | 2849.60 | 2849.5 | -0.1 | 2849.45 | -0.15 |
(8,6,3) | 6.3 | C114H190N6O83 | 2995.74 | 2995.5 | -0.2 | 2995.61 | -0.13 |
(9,7,0) | 7.0 | C110H183N7O81 | 2922.65 | 2922.1 | -0.6 | 2922.60 | -0.05 |
(9,7,1) | 7.1 | C116H193N7O85 | 3068.79 | 3069.1 | +0.3 | 3068.74 | -0.05 |
(9,7,2) | 7.2 | C122H203N7O89 | 3214.94 | 3214.8 | -0.1 | 3214.81 | -0.13 |
(9,7,3) | 7.3 | C128H213N7O93 | 3361.08 | 3361.2 | +0.1 | 3361.25 | +0.17 |
(9,7,4) | 7.4 | C134H223N7O97 | 3507.22 | 3507.5 | +0.3 | 3506.55 | -0.67 |
(10,8,0) | 8.0 | C124H206N8O91 | 3287.99 | 3288.4 | +0.4 | 3288.24 | +0.25 |
(10,8,1) | 8.1 | C130H216N8O95 | 3434.13 | 3434.0 | -0.1 | 3434.32 | +0.19 |
(10,8,2) | 8.2 | C136H226N8O99 | 3580.27 | 3580.9 | +0.6 | 3580.51 | +0.24 |
(10,8,3) | 8.3 | C142H236N8O103 | 3726.42 | 3727.4 | +1.0 | 3726.29 | -0.13 |
(10,8,4) | 8.4 | C148H246N8O107 | 3872.56 | 3873.1 | +0.5 | 3872.89 | +0.33 |
(11,9,0) | 9.0 | C138H229N9O101 | 3653.33 | 3653.3 | 0.0 | 3653.38 | +0.06 |
(11,9,1) | 9.1 | C144H239N9O105 | 3799.47 | 3800.3 | +0.8 | 3799.79 | +0.32 |
(11,9,2) | 9.2 | C150H249N9O109 | 3945.61 | 3945.6 | 0.0 | 3945.65 | +0.04 |
(11,9,3) | 9.3 | C156H259N9O113 | 4091.76 | 4091.7 | -0.1 | 4091.97 | +0.21 |
(11,9,4) | 9.4 | C162H269N9O117 | 4237.90 | 4238.6 | +0.7 | 4238.09 | +0.19 |
(11,9,5) | 9.5 | C168H279N9O121 | 4384.04c | 4384.1 | +0.1 | 4384.31 | +0.27 |
(12,10,0) | 10.0 | C152H252N10O111 | 4018.66 | 4019.7 | +1.0 | 4018.97 | +0.31 |
(12,10,1) | 10.1 | C158H262N10O115 | 4164.81 | 4165.3 | +0.5 | 4164.48 | -0.33 |
(12,10,2) | 10.2 | C164H272N10O119 | 4310.95 | 4311.5 | +0.6 | 4310.97 | +0.02 |
(12,10,3) | 10.3 | C170H282N10O123 | 4457.09 | 4457.3 | +0.2 | 4457.35 | +0.26 |
(12,10,4) | 10.4 | C176H292N10O127 | 4603.24 | 4603.6 | +0.4 | 4603.44 | +0.20 |
(12,10,5) | 10.5 | C182H302N10O131 | 4749.38c | 4749.2 | -0.2 | 4749.69 | +0.31 |
(13,11,1) | 11.1 | C172H285N11O125 | 4530.14 | 4531.2 | +1.1 | 4530.22 | +0.08 |
(13,11,2) | 11.2 | C178H295N11O129 | 4676.29 | 4676.6 | +0.3 | 4676.51 | +0.22 |
(13,11,3) | 11.3 | C184H305N11O133 | 4822.43 | 4822.3 | -0.1 | 4822.97 | +0.54 |
(13,11,4) | 11.4 | C190H315N11O137 | 4968.57 | 4969.2 | +0.6 | 4969.07 | +0.50 |
(13,11,5) | 11.5 | C196H325N11O141 | 5114.72c | 5115.2 | +0.5 | 5115.25 | +0.53 |
(13,11,6) | 11.6 | C202H335N11O145 | 5260.86c | 5261.1 | +0.2 | 5261.27 | +0.41 |
(14,12,1) | 12.1 | C186H308N12O135 | 4895.48 | 4895.0 | -0.5 | 4895.95 | +0.47 |
(14,12,2) | 12.2 | C192H318N12O139 | 5041.62 | 5041.9 | +0.3 | 5041.82 | +0.20 |
(14,12,3) | 12.3 | C198H328N12O143 | 5187.77 | 5186.8 | -1.0 | 5188.13 | +0.36 |
(14,12,4) | 12.4 | C204H338N12O147 | 5333.91 | 5333.8 | -0.1 | 5334.09 | +0.18 |
(14,12,5) | 12.5 | C210H348N12O151 | 5480.05c | 5479.5 | -0.6 | 5480.02 | -0.03 |
(14,12,6) | 12.6 | C216H358N12O155 | 5626.20c | 5625.0 | -1.2 | 5626.15 | -0.05 |
(15,13,2) | 13.2 | C206H341N13O149 | 5406.96 | 5405.8 | -1.2 | 5407.10 | +0.14 |
(15,13,3) | 13.3 | C212H351N13O153 | 5553.10 | 5551.5 | -1.6 | 5552.99 | -0.11 |
(15,13,4) | 13.4 | C218H361N13O157 | 5699.25 | 5697.9 | -1.4 | 5699.16 | -0.09 |
(15,13,5) | 13.5 | C224H371N13O157 | 5845.39c | 5844.0 | -1.4 | 5845.65 | +0.26 |
(15,13,6) | 13.6 | C230H381N13O165 | 5991.53c | 5989.7 | -1.8 | 5991.67 | +0.14 |
(15,13,7) | 13.7 | C236H391N13O169 | 6137.68c | 6134.9 | -2.8 | 6138.88 | +1.20 |
(16,14,2) | 14.2 | C220H364N14O159 | 5772.30 | 5773.5 | +1.2 | 5770.63 | -1.67 |
(16,14,3) | 14.3 | C226H374N14O163 | 5918.44 | 5916.1 | -2.3 | 5917.99 | -0.45 |
(16,14,4) | 14.4 | C232H384N14O167 | 6064.58 | 6063.8 | -0.8 | 6063.58 | -1.00 |
(16,14,5) | 14.5 | C238H394N14O171 | 6210.73c | 6208.7 | -2.0 | - | - |
(16,14,6) | 14.6 | C244H404N14O175 | 6356.87c | 6355.3 | -1.6 | - | - |
(16,14,7) | 14.7 | C250H414N14O179 | 6503.01c | 6501.3 | -1.7 | - | - |
(17,15,3) | 15.3 | C240H397N15O173 | 6283.78 | 6279.5 | -4.3 | - | - |
(17,15,4) | 15.4 | C246H407N15O177 | 6429.92 | 6428.1 | -1.8 | - | - |
(17,15,5) | 15.5 | C252H417N15O181 | 6576.07c | 6573.7 | -2.4 | - | - |
(17,15,6) | 15.6 | C258H427N15O185 | 6722.21c | 6718.5 | -3.7 | - | - |
(17,15,8) | 15.8 | C270H447N15O193 | 7014.49c | 7010.5 | -3.9 | - | - |
(18,16,3) | 16.3 | C254H420N16O183 | 6649.12 | 6646.5 | -2.6 | - | - |
(18,16,5) | 16.5 | C266H440N16O191 | 6941.40c | 6939.3 | -2.1 | - | - |
(18,16,6) | 16.6 | C272H450N16O195 | 7087.55c | 7081.9 | -5.7 | - | - |
n = 62d | [sigma] = 1.4d | n = 53d | [sigma] = 0.44d | ||||
Accuracy: | 0.02-0.08% | 0.007-0.02% |
The ceramide portion
The molecular masses of the ceramides (Mcer) were determined from the mass difference between intact PGCs (MPGC) and the released oligosaccharides (Moligos), Mcer = MPGC - Moligos + H, (Table VIII). The determined value of the most abundant ceramide of fraction 1, 632.1±1.6 Da in linear mode and 632.5 ± 0.6 Da in reflectron mode, was close to the calculated mean value 632.11 Da for d18:1-24:0 and d18:1-24:1. The mass of the less abundant ceramide was determined to 604.6 ± 1.6 Da and 605.0 ± 0.6 Da in linear and reflectron mode, respectively. The calculated value for the ceramide d18:1-22:0 was 605.06 Da.
The most abundant ceramide of fraction 2 was determined to 631.8 ± 1.2 Da in linear mode, which was consistent with the calculated mean value of 632.11 Da for d18:1-24:0 and d18:1-24:1. The less abundant ceramide was determined to 604.1 ± 1.7 Da and the calculated value for d18:1-22:0 was 605.06 Da (Table VIII).MALDI-TOF MS fingerprinting of complex mixtures of PGCs was shown in this paper to be superior to other MS techniques. Fast atom bombardment (FAB) MS has earlier been used on permethylated high-mass glycosphingolipids (>5000 Da), and molecular ions were recorded (Hanfland et al., 1981; Hanfland et al., 1984; Egge et al., 1985; Levery et al., 1989). However, FAB MS suffers from lack of sensitivity for high-mass molecular ions due to the extensive fragmentation at the hexosamine residues of permethylated glycolipids with repeating N-acetyllactosamine units. As mentioned above electrospray MS has been used for the analysis of a synthetic 25-sugar saccharide but the method is insufficient for complex mixtures as those handled here. In the present work, PGCs were isolated from human erythrocytes and separated into two fractions, 1 and 2, by silica gel chromatography. Two oligosaccharide fractions, A and B, were obtained by ceramide glycanase hydrolysis of the total mixture of PGCs followed by HPAEC. Fraction 1 and A contained mainly neutral components while fraction 2 and B were composed of sialylated species. The MALDI mass spectrum of fraction 1 showed great similarity to the spectrum of the released oligosaccharides of fraction A. In both cases [M+Na]+ ions and oligosaccharide series with increasing degree of fucosylation were detected. In positive ion linear mode, series from (6,4,1)Cer up to (17,15,6)Cer were observed for fraction 1, whereas from (6,4,0) up to (18,16,6) were found for fraction A. The molecular mass distribution envelope was centered around (10,8,3)Cer and (10,8,4)Cer in fraction 1, whereas fraction A was centered around (11,9,3) and (11,9,4). A conformity was observed between the MALDI spectra of sialylated species in fraction 2 and the released oligosaccharides in fraction B. In the negative ion linear mode, series from (7,5,0,1)Cer up to (19,17,4,1)Cer and from (8,6,0,1) up to (18,16,6,1) were detected for [M-H]- ions of fraction 2 and B, respectively. The molecular mass distribution envelope was in both cases centered around the saccharide composition (11,9,2) and (11,9,3). The mass difference between the intact PGCs and the released oligosaccharides with identical sugar composition fits exactly with the ceramide mass. This is conclusive evidence for the existence of such large glycolipids in human erythrocytes. In addition, MALDI-TOF MS revealed a microheterogeneity of these large glycolipids which is far beyond what could be detected by earlier used methods.
The different constituent monosaccharides and their linkages were concluded from methylation analysis of human erythrocyte PGCs (Koscielak et al., 1976; Miller-Podraza et al., 1993). NeuAc-, Fuc-, Gal-, -2Gal-, -3Gal-, -3,6Gal- and -4GlcNAc- were found, which suggested a branched N-acetyllactosamine core with terminals of Gal, sialic acid and fucose. The high degree of fucosylation of some PGC species revealed by the MALDI MS is in line with branched structures. Saccharides characterized by x/y < 2 (seen only in series with odd number of HexNAcs, e.g., in (11,9,5) or (13,11,6) of fraction A) may be branched at every N-acetyllactosamine unit since Fuc in the investigated PGCs occurs primarily as terminal sugar in H-blood group determinants. The PGCs also contain Lewis X and Y determinants with Fuc[alpha]3 linked to GlcNAc detectable by specific monoclonal antibodies; however, the 1,3,4-substituted GlcNAc occurs in mixtures of PGCs only in trace amounts as shown by analysis of partially methylated alditol acetates (not shown here). Less fucosylated structures could be explained by elongations without branching or by the presence of terminal Gal residues in a fully branched core. Thus, the series with x/y [ge] 2 could be explained by one or more positions lacking a branch anywhere in the core structure and having a repeated N-acetyllactosamine in linear sequence. In fact, we have evidence that human erythrocyte PGCs are heterogeneous both concerning substitutions and degree of branching. Degradation with endo-[beta]-galactosidase not expected to cleave between branches spaced by only one N-acetyllactosamine, produces series of ceramide-linked and free oligosaccharides, whose composition agrees with cleavage of internal parts of poly-N-acetyllactosamine core chains (Miller-Podraza et al., unpublished observations). This indicates that the enzyme adding GlcNAc in 6 position during the biosynthesis of human red cell PGCs is of insufficient activity to produce a fully branched core. This differs from rabbit red cells, where the PGCs were regularly branched and fully terminated with Gal[alpha]3 (Dabrowski et al., 1988). Interpretation of MALDI-TOF spectra of endo-[beta]-galactosidase degradation products of human erythrocyte PGCs will be discussed elsewhere. Recent studies on the enzymatic synthesis of branched polylactosaminoglycans using enzymes from human embryonal carcinoma cells presented evidence that 6-linked branches may be added midchain to linear 3-linked N-acetyllactosamine chains (Leppänen et al., 1997, 1998). The proposal was that branched structures like PGCs are synthesized from preformed linear sequences of N-acetyllactosamines coupled in 3-linkages by a [beta]1,6-N-acetylglucosaminyltransferase which is able to add GlcNAc on any Gal along the chain. If this enzyme is not active enough, the result would be insufficient branching and sensitivity to endo-[beta]-galactosidase as found for PGCs. This incomplete branching and the successive extension with N-acetyllactosamine combined with the varying substitution with fucose and sialic acid produces a high degree of microheterogeneity, which is reflected by the unresolved extended interval on the thin-layer chromatogram (FigureTable VII.
Structured | In Fig.e | Formula | CMb [M-H]- | OM (linear) [M-H]- | [Delta] (linear) | OM (reflectron) [M-H]- | [Delta] (reflectron) |
(8,6,0,1) | 6.0 | C107H177N7O79 | 2824.57 | 2825.2 | +0.6 | 2824.18 | -0.39 |
(8,6,1,1) | 6.1 | C113H187N7O83 | 2970.72 | 2972.1 | +1.4 | 2971.13 | +0.42 |
(8,6,2,1) | 6.2 | C119H197N7O87 | 3116.86c | 3117.8 | +0.9 | 3115.80 | -0.35 |
(9,7,0,1) | 7.0 | C121H200N8O89 | 3189.91 | 3190.3 | +0.4 | 3189.50 | -0.41 |
(9,7,1,1) | 7.1 | C127H210N8O93 | 3336.05 | 3336.0 | 0.0 | 3336.33 | +0.28 |
(9,7,2,1) | 7.2 | C133H220N8O97 | 3482.20c | 3482.5 | +0.3 | 3482.47 | +0.27 |
(9,7,3,1) | 7.3 | C139H230N8O101 | 3628.34c | 3629.4 | +1.1 | 3628.49 | +0.15 |
(10,8,0,1) | 8.0 | C135H223N9O99 | 3555.25 | 3555.5 | +0.3 | 3555.41 | +0.16 |
(10,8,1,1) | 8.1 | C141H233N9O103 | 3701.39 | 3702.2 | +0.8 | 3701.12 | -0.27 |
(10,8,2,1) | 8.2 | C147H243N9O107 | 3847.53c | 3848.2 | +0.7 | 3847.53 | 0.00 |
(10,8,3,1) | 8.3 | C153H253N9O111 | 3993.68c | 3994.2 | +0.5 | 3993.59 | -0.09 |
(11,9,0,1) | 9.0 | C149H246N10O109 | 3920.59 | 3920.4 | -0.2 | 3920.56 | -0.03 |
(11,9,1,1) | 9.1 | C155H256N10O113 | 4066.73 | 4067.8 | +1.1 | 4066.54 | -0.19 |
(11,9,2,1) | 9.2 | C161H266N10O117 | 4212.87c | 4213.1 | +0.2 | 4212.75 | -0.12 |
(11,9,3,1) | 9.3 | C167H276N10O121 | 4359.02c | 4359.0 | 0.0 | 4358.91 | -0.11 |
(11,9,4,1) | 9.4 | C173H286N10O125 | 4505.16c | 4505.5 | +0.3 | 4504.60 | -0.56 |
(12,10,0,1) | 10.0 | C163H269N11O119 | 4285.92 | 4286.7 | +0.8 | 4285.67 | -0.25 |
(12,10,1,1) | 10.1 | C169H279N11O123 | 4432.07 | 4432.0 | -0.1 | 4432.26 | +0.19 |
(12,10,2,1) | 10.2 | C175H289N11O127 | 4578.21c | 4577.6 | -0.6 | 4577.79 | -0.42 |
(12,10,3,1) | 10.3 | C181H299N11O131 | 4724.35c | 4724.7 | +0.4 | 4724.04 | -0.31 |
(12,10,4,1) | 10.4 | C187H309N11O135 | 4870.50c | 4869.9 | -0.6 | 4869.77 | -0.72 |
(13,11,0,1) | 11.0 | C177H292N12O129 | 4651.26c | 4652.4 | +1.1 | 4651.17 | -0.09 |
(13,11,1,1) | 11.1 | C183H302N12O133 | 4797.40 | 4798.6 | +1.2 | 4796.70 | -0.70 |
(13,11,2,1) | 11.2 | C189H312N12O137 | 4943.55c | 4943.9 | +0.4 | 4942.82 | -0.73 |
(13,11,3,1) | 11.3 | C195H322N12O141 | 5089.69c | 5089.3 | -0.4 | 5088.69 | -1.00 |
(13,11,4,1) | 11.4 | C201H332N12O145 | 5235.83c | 5235.8 | 0.0 | 5235.31 | -0.52 |
(13,11,5,1) | 11.5 | C207H342N12O149 | 5381.98c | 5381.2 | -0.8 | 5381.97 | -0.01 |
(14,12,0,1) | 12.0 | C191H315N13O139 | 5016.60 | 5016.4 | -0.2 | 5016.01 | -0.59 |
(14,12,1,1) | 12.1 | C197H325N13O143 | 5162.74 | 5162.9 | +0.2 | 5162.31 | -0.43 |
(14,12,2,1) | 12.2 | C203H335N13O147 | 5308.88c | 5309.3 | +0.4 | 5307.59 | -1.29 |
(14,12,3,1) | 12.3 | C209H345N13O151 | 5455.03c | 5454.6 | -0.4 | 5454.34 | -0.69 |
(14,12,4,1) | 12.4 | C215H355N13O155 | 5601.17c | 5600.7 | -0.5 | 5601.36 | +0.19 |
(14,12,5,1) | 12.5 | C221H365N13O159 | 5747.31c | 5747.6 | +0.3 | 5746.45 | -0.86 |
(15,13,1,1) | 13.1 | C211H348N14O153 | 5528.08 | 5526.3 | -1.8 | 5527.56 | -0.52 |
(15,13,2,1) | 13.2 | C217H358N14O157 | 5674.22c | 5674.5 | +0.3 | 5673.21 | -1.01 |
(15,13,3,1) | 13.3 | C223H368N14O161 | 5820.36c | 5819.0 | -1.4 | 5819.77 | -0.59 |
(15,13,4,1) | 13.4 | C229H378N14O165 | 5966.51c | 5964.2 | -2.3 | 5967.24 | +0.74 |
(15,13,5,1) | 13.5 | C235H388N14O169 | 6112.65c | 6111.2 | -1.5 | 6113.04 | +0.39 |
(15,13,6,1) | 13.6 | C241H398N14O173 | 6258.79c | 6256.5 | -2.3 | 6258.51 | -0.28 |
(16,14,1,1) | 14.1 | C225H371N15O163 | 5893.42 | 5895.3 | +1.9 | - | - |
(16,14,2,1) | 14.2 | C231H381N15O167 | 6039.56c | 6038.1 | -1.5 | - | - |
(16,14,3,1) | 14.3 | C237H391N15O171 | 6185.70c | 6187.1 | +1.4 | 6184.28 | -1.42 |
(16,14,4,1) | 14.4 | C243H401N15O175 | 6331.85c | 6331.0 | -0.9 | 6331.91 | +0.06 |
(16,14,5,1) | 14.5 | C249H411N15O179 | 6477.99c | 6476.5 | -1.5 | - | - |
(16,14,6,1) | 14.6 | C255H421N15O183 | 6624.13c | 6623.7 | -0.4 | 6625.48 | +1.35 |
(17,15,2,1) | 15.2 | C245H404N16O177 | 6404.90c | 6402.4 | -2.5 | - | - |
(17,15,4,1) | 15.4 | C257H424N16O185 | 6697.18c | 6696.0 | -1.2 | - | - |
(17,15,5,1) | 15.5 | C263H434N16O189 | 6843.33c | 6842.8 | -0.5 | 6843.76 | +0.43 |
(17,15,6,1) | 15.6 | C269H444N16O193 | 6989.47c | 6989.1 | -0.4 | 6991.69 | +2.22 |
(17,15,7,1) | 15.7 | C275H454N16O197 | 7135.61c | 7133.3 | -2.3 | - | - |
(18,16,2,1) | 16.2 | C259H427N17O187 | 6770.23c | 6769.0 | -1.2 | - | - |
(18,16,3,1) | 16.3 | C265H437N17O191 | 6916.38c | 6915.8 | -0.6 | - | - |
(18,16,4,1) | 16.4 | C271H447N17O195 | 7062.52c | 7059.2 | -3.3 | - | - |
(18,16,5,1) | 16.5 | C277H457N17O199 | 7208.66c | 7206.6 | -2.1 | - | - |
(18,16,6,1) | 16.6 | C283H467N17O203 | 7354.81c | 7351.8 | -3.0 | - | - |
n = 55n | [sigma] = 1.2n | n = 44n | [sigma] = 0.65n | ||||
Accuracy: | 0.02-0.04% | 0.009-0.02% |
Table VIII.
Fraction and mode | Mcer (Da) measured | Mcer calc. mass (Da) | Ceramide | Formula |
Fraction 1, linear | 632.1±1.6, n = 36b | |||
Fraction 1, reflectron | 632.5±0.6, n = 35b | 632.11 | d18:1-24:0/1 | C42H82(80)NO2 |
Fraction 2, linear | 631.8±1.2, n = 36b | |||
Fraction 1, linear | 604.6±1.6, n = 12b | |||
Fraction 1, reflectron | 605.0±0.6, n = 13b | 605.06 | d18:1-22:0 | C40H78NO2 |
Fraction 2 linear | 604.1±1.7, n = 23b |
Figure 4. MALDI-TOF mass spectrum of fraction A in (A) positive ion linear mode and (B) positive ion reflectron mode. Annotations as HexNAc.Fuc (Table VI).
Figure 5. MALDI-TOF mass spectrum of fraction B in (A) negative ion linear mode and (B) negative ion reflectron mode. Annotations as HexNAc.Fuc (Table VII).
From these results, nothing can be concluded on the binding epitope for H.pylori. However, MALDI-TOF MS technique will be helpful in characterizing the structure of partial degradation products, which may retain binding activity. The binding of the bacterium is sialic acid dependent; removal of sialic acid by acid or by neuraminidase completely abolishes binding, and the same result is obtained after mild periodate oxidation, which eliminates only C-9, or C-8 and C-9, of the glycerol tail of sialic acid (Miller-Podraza et al., 1996). However, animal PGCs, which also contain sialic acid, are inactive (Miller-Podraza, et al., 1997c). The specificity for human PGCs may reside in an epitope conformation where sialic acid interacts with hydrogen bonds between C-9 of its glycerol tail and an incompletely branched N-acetyllactosamine core (Karlsson, 1998). Glycosphingolipid material
Bovine brain gangliosides (mixture of GM1, GD1a, GD1b, and GT1b) were purchased from Calbiochem (La Jolla, CA, USA). PGCs of human erythrocyte membranes, blood group O, were prepared in our laboratory according to the peracetylation procedure as described (Miller-Podraza et al., 1993). The main fraction obtained after Sephadex LH 60 chromatography was purified by silica gel chromatography (silica gel suspended in chloroform) using chloroform/methanol 3:1 as elution solvent. The separation resulted in two polyglycosylceramide fractions differing in sialic acid content (see Table 1). Colorimetric assays
Hexose, sialic acid and sphingosine were assayed according to references given earlier (Miller-Podraza et al., 1993). Binding assay
H.pylori, CCGU 17874 strain, was obtained from Culture Collection of Göteborg University (CCGU). Cultivation in Ham's F12 liquid medium and overlay of TLC plates with 35S-labeled cells were performed as described earlier (Karlsson and Strömberg, 1987; Miller-Podraza et al., 1996). Hydrolysis by ceramide glycanase and separation of oligosaccharides
Digestion of PGCs by ceramide glycanase (Rhodococcus) was performed as described (Miller-Podraza et al., 1997c). The products were separated by phase partition in chloroform/methanol/water, 10:5:3 (Li and Li, 1989) and the carbohydrates contained in the upper phase were purified by Sephadex G-15 chromatography. The saccharide mixture was separated by HPAEC as described (Miller-Podraza et al., 1993). MALDI-TOF MS
MALDI mass spectra were obtained on a TofSpec-E time-of-flight mass spectrometer (Micromass, Manchester, England) equipped with delayed extraction (DE) and a nitrogen laser (337 nm, 4 ns pulse, LSI, Boston, MA) operated in either the reflectron or linear mode. The accelerating voltage used was 20 kV in reflectron mode and 25 kV in linear mode. For delayed extraction a 2 kV potential difference between the probe and the extraction lens was applied with a time delay of 600 ns after each laser pulse. The molecular ions, [M+H]+, of the ACTH clip 18-39 at m/z = 2466.7 (average mass) and bovine insulin at m/z = 5734.5 (average mass) were used in positive ion mode for external mass calibration. The corresponding [M-H]- ions were used for calibration in the negative ion mode. As matrix 2,5-dihydroxybenzoic acid (about 15 mg/ml) in water was used for all samples and calibration compounds. Equal volumes (5 µl) of the sample and matrix solutions were mixed from which 1 µl was applied on the stainless steel target. The mixture was allowed to dry at ambient temperature before introduction into the mass spectrometer. The neutral glycolipids and oligosaccharides produced sodium adduct ions, [M+Na]+, and were detected in positive ion mode. The sialic acid containing components produced [M-H]- ions, which were detected in negative ion mode. Approximately 200-300 shots were accumulated per spectrum. The pressure in the TOF analyzer was 1 × 10-7 mbar. Although the delayed extraction data obtained in reflectron mode was isotopically resolved in the lower mass range (below 3000 amu) all spectra were smoothed to receive an average mass in order to simplify the data treatment.
MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Hex, hexose; HexNAc, N-acetylhexosamine; Fuc, fucose; NeuAc, N-acetylneuraminic acid; PGCs, polyglycosylceramides. Polysaccharides are denoted in the text according to number of Hex, HexNAc, Fuc, and NeuAc units, respectively; e.g., (8,6,0,1) means that the molecule contains totally 15 monosaccharides. For ceramide abbreviations, d18:1 means sphingosine (1,3-dihydroxy-2-amino-4-trans-octadecane) and, e.g., 24:1 means a nonhydroxy fatty acid with 24 carbon atoms and one double bond.
Materials and methods
Abbreviations
References
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