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Nature Methods
Shotgun glycomics: a microarray strategy for functional
glycomics Xuezheng Song, Yi Lasanajak, Baoyun Xia, Jamie Heimburg-Molinaro, Jeanne M Rhea, Hong Ju, Chunmei Zhao, Ross J Molinaro, Richard D Cummings & David F Smith Supplementary Figure 1 The bifunctional fluorescent derivatization of glycosphingolipids and the
validation of microarray printing.
Supplementary Figure 2 Thin-layer chromatography (TLC) image of GM1, GD1a, GT1b, BBG and
their corresponding AOAB conjugates.
Supplementary Figure 3 The two-dimensional HPLC separation of BBG GSL-AOABs.
Supplementary Figure 4 The elucidation of the structure of fraction 12.
Supplementary Figure 5 Fluorescent AOAB derivatization of PC3 cells GSLs.
Supplementary Figure 6 The lectin binding to PC3 cell glycosphingolipids microarray.
Supplementary Table 1 HPLC and Mass spectrometry characterization of BBG two-dimensional
fractions.
Supplementary Table 2 Raw data of antibody binding from control sera and sera from individuals
with Lyme disease.
Supplementary Table 3 MALDI-TOF MS and MS/MS data of the 10 peaks collected from C18 HPLC
separation of PC3 cell GSL-AOABs.
Nature Methods: doi: 10.1038/nmeth.1540
Supplementary Figure 1. The bifunctional fluorescent derivatization of glycosphingolipids and the validation of microarray printing. (a) The monosialyl ganglioside GM1 (Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-ceramide) is treated with ozone, PNPA, and diamines with different lengths. Treatment of PNPA conjugates with ethylenediamine yielded AEAB
1Nature Methods: doi: 10.1038/nmeth.1540
conjugates, while treatment with 1,4-diaminobutane and 1,8-diaminooctane (ODA) generated homologs with varied lengths, termed N-(aminobutyl)-2-amino benzamide (ABAB) and N-(aminooctyl)-2-amino benzamide (AOAB) conjugates. The fluorescent GM1 conjugates are named as GM1-AEAB, GM1-ABAB and GM1-AOAB; (b) C18-HPLC profiles of GM1-AEAB, GM1-ABAB and GM1-AOAB; all products gave a single fluorescent product peak by C18-HPLC; (c) MALDI-TOF profiles of GM1-AEAB, GM1-ABAB and GM1-AOAB, showing masses matching calculated values. (d) The test printing of GM1-AEAB, GM1-ABAB and GM1-AOAB at different concentrations. The three GM1 derivatives were quantified based on fluorescence, printed on three different glass slides, and interrogated with biotinylated cholera toxin subunit B (CTSB), which specifically binds the GM1 glycan. They were printed on 4 slides: nitrocellulose-vendor 1, nitrocellulose-vendor 2, NHS and epoxy slides. For each sample, 8 concentrations (200, 100, 50, 25, 12.5, 6.25, 3.13, 1.6 µM) were printed in replicates of n = 4 for each concentration. The slides were interrogated with biotinylated CTSB and detected by cyanine5-streptavidin. The slides were scanned at different photomultiplier tube (PMT) settings. PMT 70 has higher detecting sensitivity than PMT 50. For nitrocellulose slides, lower PMT (50) had to be used due to the autofluorescence of nitrocellulose membrane. For glass slides, either NHS or epoxy, higher PMT (70) was used to reach a higher sensitivity. When background is high (such as the epoxy slide in the following picture), lower PMT (50) can be used to reduce the background. The pictures show the fluorescent images on different slides and their brightness/contrast ratio have been adjusted. The comparative studies by printing of the three GM1 derivatives on nitrocellulose slides and NHS- or epoxy-slides demonstrate that the N-(aminooctyl)-2-amino benzamide (AOAB) conjugates, GM1-AOAB with the C8 extension was detected by CTSB with greater sensitivity than the other derivatives. The longer alkyl chain of GM1-AOAB, which increases its hydrophobicity, may increase the printing efficiency or, more possibly, may help to organize the parallel fatty acid chain, reducing its interference between binding of CTSB and glycan.
2Nature Methods: doi: 10.1038/nmeth.1540
Supplementary Figure 2. Thin-layer chromatography (TLC) image of GM1, GD1a, GT1b, BBG, and their corresponding AOAB conjugates by orcinol staining (a) or fluorescence upon UV irradiation (b).
3Nature Methods: doi: 10.1038/nmeth.1540
Supplementary Figure 3. The 2D HPLC separation of BBG GSL-AOABs. Bovine brain gangliosides were treated with ozone, quenched with methyl sulfide, dried over nitrogen and conjugated with PNPA. The mixture was precipitated with 10-20 volumes of water, centrifuged and filtered through a 0.2 µm membrane. The precipitate, unreacted PNPA, was discarded. The filtrate was lyophilized and treated with 1,8-diaminooctane, dried, neutralized with acetic acid. (a) The crude product was injected onto a C18 semi-prep HPLC column and eluent from 41-54
4Nature Methods: doi: 10.1038/nmeth.1540
minutes (the gray area) was pooled as the fluorescently labeled glycolipids fractions. A Vydac C18 column (250 mm x 9.2 mm) was used. The mobile phase was acetonitrile and water with 0.1% trifluoroacetic acid (TFA). The flow rate was 2.5 ml min-1. The concentration of acetonitrile increased from 15% to 90% linearly over 75 minutes. (b) The pooled eluent was dried, lyophilized, reconstituted in methanol, and injected into normal phase semi-preparative HPLC for 1st dimension separation; 20 fractions were collected. (c) All normal phase fractions were briefly evaporated in Speed-vac to remove organic solvent and then lyophilized. The lyophilized material was reconstituted in water and separated on a C18 reverse phase HPLC column. Fractions were collected manually as peaks. The HPLC profiles (retention time 15 to 28 minutes) of the second dimension C18 HPLC separation of the normal fractions are shown below (from bottom to top: normal phase fraction #1 to #20). Fractions were rechromatographed on the same C18 HPLC column with a different gradient to check the homogeneity and further separate glycans if several peaks were present. A total of 40 fractions were obtained in the tagged ganglioside library.
5Nature Methods: doi: 10.1038/nmeth.1540
Supplementary Figure 4. The elucidation of the structure of fraction 12. Water loss can occur during MALDI analysis, however the loss of water can also occur prior to MALDI analysis as in a lactone or anhydrosugar formation. We offer multiple lines of evidence to support GD1b-lactone as the structure of fraction 12. These lines of evidence include: 1) C18 HPLC and MS analyses, which showed that upon storage this fraction separated into 2 major components with Peak 2 being the original component (the lactone) and Peak 1 being the hydrolyzed product from the lactone; 2) Neuraminidase treatment, showing resistance to neuraminidase as expected for a lactone; 3) Chemical treatment, since lactones are prone to amidation with amine while the corresponding disialyl GSLs and anhydrosugars are not. Based on these data, we are confident that the structure is a GD1b-lactone conjugate. (a) The C18-HPLC profiles of fraction #12 after ~1 year at -20°C storage (with several freeze-thaw cycles), Peak 1 (20.01 min) and Peak 2 (20.44 min) are annotated. The two peaks were isolated from HPLC and analyzed by mass spectrometry; (b) The MALDI-TOF spectra of Peak 1 (top panel), Peak 2 (middle panel), and the
6Nature Methods: doi: 10.1038/nmeth.1540
MALDI-TOF/TOF spectrum of Peak 2 (bottom panel). Peak 1 showed a dominant molecular ion at m/z 1905 [M+H]+, matching (Hex3HexNAc1Neu5Ac2) without loss of water. It is reasonable to conclude that peak 1 is formed by hydrolysis of the proposed lactone structure (Peak 2), which is consistent with Peak 2’s longer retention time (greater hydrophobicity). (c) The C18-HPLC profiles of three fractions before and after neuraminidase treatment; Both GD1a-AOAB (fraction 24) and GD1b-AOAB (Peak 1 in fraction 12) were digested to GM1-AOAB (fraction 9) but the GD1b-lactone-AOAB (Peak 2 in fraction 12) is resistant to neuraminidase; (d) Ethylenediamine treatment of fraction 9 (GM1-AOAB) as a control and fraction 12. A new peak X is generated from GD1b-lactone-AOAB while GM1-AOAB and GD1b-AOAB are not affected. (e) The reaction scheme of GD1b-lactone-AOAB with ethylenediamine. The new peak X is isolated from HPLC and characterized by MALDI-TOF (top panel) and MALDI-TOF/TOF (bottom panel), which confirmed the expected molecular weight and fragmentation pattern of the amide of GD1b-AOAB.
7Nature Methods: doi: 10.1038/nmeth.1540
Supplementary Figure 5. Fluorescent AOAB derivatization of PC3 cells GSLs. (a) C18-HPLC profile of the GSL-AOAB derivatives from a glycolipid extract of PC3 cells; (b) The MS analysis of fraction 16 and its tentative structure. In negative mode (top panel), this fraction showed only a single peak at 1535.1[M-H]-, suggesting an absolute mass of 1536. Interestingly, in positive mode, this fraction showed dominant peaks at 1456.8[M+H]+ and 1478.8[M+Na]+ (middle panel), with no detection at expected 1537[M+H]+. The striking discrepancy of the MS spectra in positive and negative mode clearly suggested a labile moiety with a mass of 80. While both sulfate and phosphate can give an 80 mass shift, it is known that de-sulfation is common while glycan phosphates are quite stable to fragmentation during MALDI-TOF analysis. Furthermore, the HPLC profile shift upon mild acid hydrolysis and resistance to alkaline phosphatase strongly suggests that the glycan associated with fraction 16 is sulfated. The 1456.8[M+H]+ molecular ion matches well with the composition of Hex4HexNAc1 possessing a C16-N-acyl sphingosine moiety. MS/MS analysis of peak 1456.8 (bottom panel) gave a clear linear pattern of Hex-HexNAc-Hex-Hex-Hex, suggesting a globo-series GSL structure with a sulfated pentasaccharide glycan.
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Supplementary Figure 6. The lectin binding to PC3 cell glycosphingolipids microarray. Thirty-three fractions of PC3 GSL-AOABs were separated by C18-HPLC and printed; 11 GSL-AOAB or GAEAB structures were printed on the same microarray as controls. The concentration of lectins (a) ConA; (b) MAA; (c) AAL and (d) SNA used is 10 µg ml-1.
9Nature Methods: doi: 10.1038/nmeth.1540
Supplementary Table 1. HPLC and Mass spectrometry characterization of BBG 2-dimensional fractions. (a) All 40 fractions were characterized by MALDI-TOF/TOF and the MS and MS/MS spectra are included below. The major peaks and most probable glycan compositions are summarized in the following table. These fractions represent a tagged library of bovine brain gangliosides and were printed as described in the main text. The C18-HPLC profiles of the final re-purification are also attached below the table. All of the fractions (#1-40) are denoted on the profiles. For this re-purification, a linear gradient from 60% acetonitrile in 0.1% TFA to 90% acetonitrile in 0.1% TFA in 10 minutes was used.
BBG-AOAB
2D fractions
# NP fraction # and
retention time (min)
RP fraction # and retention time
(min) RP-HPLC repurify
Observed Mass (RP) Observed Mass (RN) Glycan Composition
F-01 NP01-28.34min RP01-22.85 1248.766 1246.852 Hex2Neu5Ac1 F-02 NP02-29.09min RP03-21.60 1344.701, 1025.492 1455.824 Hex3HexNAc1 F-03 NP03-30.35min RP02-21.51 1613.943, 1823.134 1612.033 Hex3HexNAc1Neu5Ac1 F-04 NP03-30.35min RP03-22.19 1451.87 1449.924 Hex2HexNAc1Neu5Ac1 F-05 NP03-30.35min RP04-22.52 1521.884 1519.986, 1538.003 Hex2Neu5Ac2-H2O F-06 NP04-31.16min RP03-23.88 1583.895, 1793.1 1581.978 Hex3HexNAc1Neu5Ac1 F-07 NP05-31.72min RP01-21.58 1887.175 1885.145 Hex3HexNAc1Neu5Ac2-H2O F-08 NP06-32.70min RP03-21.05 1376.663 1336.779 Hex3HexNAc1Neu5Ac1 F-09 NP06-32.70min RP04-22.04 1614.03 1612.152 Hex3HexNAc1Neu5Ac1 F-10 NP07-34.76min RP01-20.27 1390.71 1366.797, 1609.876 Hex3HexNAc1Neu5Ac2 F-11 NP07-34.76min RP02-20.72 1887.057 1886.029, 1904.064 Hex3HexNAc1Neu5Ac2-H2O F-12 NP07-34.76min RP03-20.99 1887.052 1886.042 Hex3HexNAc1Neu5Ac2-H2O F-13 NP07-34.76min RP04-21.57 1613.941 1612.072 Hex3HexNAc1Neu5Ac1 F-14 NP07-34.76min RP05-22.08 1614.195 1612.127 Hex3HexNAc1Neu5Ac1 F-15 NP08-36.52min RP01-19.56 1685.771 1639.9 Hex3HexNAc1Neu5Ac2 F-16 NP08-36.52min RP03-20.74 1817.455 1815.44 Hex3HexNAc2Neu5Ac1 F-17 NP08-36.52min RP06-23.63 1509.816 1485.867 Hex3HexNAc1Neu5Ac1 F-18 NP09-38.12min RP03-20.48 1593.719 1570.049 Hex3HexNAc2Neu5Ac1 F-19 NP09-38.12min RP04-21.04 2020.099 2018.157 Hex3HexNAc3Neu5Ac1 F-20 NP09-38.12min RP06-22.65 1522.028 1520.033 Hex2Neu5Ac2-H2O F-21 NP09-38.12min RP07-23.60 1782.876, 1875.06 1740.892 Hex3HexNAc1Neu5Ac2 F-22 NP10-38.49min RP08-23.51 1874.979 1895.103 Hex3HexNAc1Neu5Ac2 F-23 NP11-40.46min RP02-20.30 2108.3 2128.412 Hex3HexNAc2Neu5Ac2 F-24 NP11-40.46min RP03-21.78 1905.15 1885.225 Hex3HexNAc1Neu5Ac2
1Nature Methods: doi: 10.1038/nmeth.1540
2
F-25 NP11-40.46min RP04-23.50 1629.864 2208.283 Hex3HexNAc1Neu5Ac3-H2O F-26 NP12-42.3min RP01-20.23 1681.946, 1756.127 1640.054 Hex3HexNAc1Neu5Ac2 F-27 NP12-42.3min RP02-20.70 a 2178.554, 2196.576 2176.605 Hex3HexNAc1Neu5Ac3-H2O F-28 NP12-42.3min RP02-20.70 b 2178.255 2176.523 Hex3HexNAc1Neu5Ac3-H2O F-29 NP12-42.3min RP02-20.70 c 1887.030, 2178.135 2094.3 Hex3HexNAc1Neu5Ac3-H2O F-30 NP12-42.3min RP03-21.60 1404.58 1380.755 Hex3HexNAc1Neu5Ac1 F-31 NP14-46.4min RP06-23.52 1801.09 1758.933 Hex3HexNAc1Neu5Ac2 F-32 NP17-50.83min RP01-20.37 a 2193.972, 2212.007 2210.433 Hex3HexNAc1Neu5Ac2Neu5Gc
F-33 NP17-50.83min RP01-20.37 b 2194.168 2214.479 Hex3HexNAc1Neu5Ac2Neu5Gc-
H2O F-34 NP18-52.88min RP02-19.91 a 2178.128 2194.29 Hex3HexNAc1Neu5Ac3-H2O F-35 NP18-52.88min RP02-19.91 b 2178.224 2176.365 Hex3HexNAc1Neu5Ac3-H2O F-36 NP18-52.88min RP03-20.43 a 2177.943 2198.369 Hex3HexNAc1Neu5Ac3-H2O F-37 NP18-52.88min RP03-20.43 b 2178.492 2198.499 Hex3HexNAc1Neu5Ac3-H2O F-38 NP19-55.00min RP01-20.20 a 2451.852 2467.703 Hex3HexNAc1Neu5Ac4-2H2O F-39 NP19-55.00min RP01-20.20 b 2178.612 2467.557 Hex3HexNAc1Neu5Ac4-2H2O F-40 NP20-57~69min RP01-21.36 1695.854 1654.123 Hex3HexNAc1Neu5Ac2
Nature Methods: doi: 10.1038/nmeth.1540
(b) Re-chromatography of all 40 HPLC fractions on C18 HPLC. F-01
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F-02
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F-06
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RF-10AxlCalbiochem-BBG-PO-NP04-RP03
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F-11
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F-16
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F-19
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F-21
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F-26
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F-27, 28, 29 (left, middle and right peaks respectively)
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F-30
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F-31
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F-32, 33 (left and right peaks respectively)
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F-34, 35 (left and right peaks respectively)
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F-36, 37 (left and right peaks respectively)
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F-38, 39 (left and right peaks respectively)
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F-40
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(c) MS and MS/MS spectra of the 40 fractions. Fraction 01
1248.766
957.6261457.946
2009-11-10-BBG-PO-LIFT\0_I5\1\1SRef
0.0
0.5
1.0
1.5
4x10
Inte
ns. [
a.u.
]
633.201
957.445
1248.841795.277370.093
2009-11-10-BBG-PO-LIFT\0_I5\1\1248.7660.LIFT\1SRef
0.0
0.5
1.0
1.5
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 02 1025.492
734.352 1344.701 2476.8302231.739
2009-11-10-BBG-PO-LIFT\0_I7\1\1SRef
0
2000
4000
6000
8000
Inte
ns. [
a.u.
]
633.770
1320.783
957.113370.087795.465 1159.549488.961
2009-11-10-BBG-PO-LIFT\0_I7\1\1344.7006.LIFT\1SRef
0
2000
4000
6000
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 03 1613.943
1823.134
1298.674
2009-11-10-BBG-PO-LIFT\0_I9\1\1SRef
0
1000
2000
3000
4000
Inte
ns. [
a.u.
]
632.733
1322.181 1614.722794.638365.532
2009-11-10-BBG-PO-LIFT\0_I9\1\1613.9428.LIFT\1SRef
0
1000
2000
3000
4000
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
10Nature Methods: doi: 10.1038/nmeth.1540
Fraction 04 1451.870
1204.185795.571 1661.029
2009-11-10-BBG-PO-LIFT\0_I11\1\1SRef
0.0
0.5
1.0
1.5
2.04x10
Inte
ns. [
a.u.
]
633.359
1160.748
795.364 1451.989204.050 489.230
2009-11-10-BBG-PO-LIFT\0_I11\1\1451.8699.LIFT\1SRef
0
1
2
3
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 05 1521.884
957.6601248.792795.573 1731.056
2009-11-10-BBG-PO-LIFT\0_K1\1\1SRef
0.0
0.5
1.0
1.5
2.0
4x10
Inte
ns. [
a.u.
]
633.276
957.371
795.285 1522.004489.162 1230.720205.992
2009-11-10-BBG-PO-LIFT\0_K1\1\1521.8839.LIFT\1SRef
0
1
2
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 06
1583.895
1793.1001314.745
2009-11-10-BBG-PO-LIFT\0_K3\1\1SRef
0.0
0.5
1.0
1.5
4x10
Inte
ns. [
a.u.
]
603.079
1292.585
765.050365.791 1585.2511157.977
2009-11-10-BBG-PO-LIFT\0_K3\1\1583.8953.LIFT\1SRef
0
1
2
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
11Nature Methods: doi: 10.1038/nmeth.1540
Fraction 07 1887.175
1614.061
2096.376
2009-11-10-BBG-PO-LIFT\0_K5\1\1SRef
0
2000
4000
6000
Inte
ns. [
a.u.
]
632.627
1596.831956.687179.703 435.387 1885.6301248.842
2009-11-10-BBG-PO-LIFT\0_K5\1\1887.1747.LIFT\1SRef
0.0
0.5
1.0
1.5
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 08 1376.663
1085.5321886.987
2009-11-10-BBG-PO-LIFT\0_K7\1\1SRef
0
1000
2000
Inte
ns. [
a.u.
]
1069.071
1359.312387.956 704.184 951.204
2009-11-10-BBG-PO-LIFT\0_K7\1\1376.6634.LIFT\1SRef
0.0
0.5
1.0
1.5
2.04x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 09 1614.030
1365.964 1823.219795.610
2009-11-10-BBG-PO-LIFT\0_K9\1\1SRef
0.0
0.5
1.0
1.5
2.0
4x10
Inte
ns. [
a.u.
]
633.010
1322.5541615.553
794.970365.722 1160.227
2009-11-10-BBG-PO-LIFT\0_K9\1\1614.0299.LIFT\1SRef
0.0
0.5
1.0
1.5
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
12Nature Methods: doi: 10.1038/nmeth.1540
Fraction 10 1390.710
1633.7681099.589
2178.100
2009-11-10-BBG-PO-LIFT\0_K11\1\1SRef
0
1000
2000
3000
4000
Inte
ns. [
a.u.
]
1100.040
1361.067
2009-11-10-BBG-PO-LIFT\0_K11\1\1390.7103.LIFT\1SRef
0
500
1000
1500
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 11 1887.057
2031.2171322.8471613.951795.580 2240.400
2009-11-10-BBG-PO-LIFT\0_K13\1\1SRef
0
1000
2000
3000
4000
5000
Inte
ns. [
a.u.
]
632.652
1321.765794.608365.481 1886.5631520.9241159.575
2009-11-10-BBG-PO-LIFT\0_K13\1\1887.0572.LIFT\1SRef
0.0
0.5
1.0
1.5
2.0
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 12 1887.052
2096.2451344.762795.569 1613.930
2009-11-10-BBG-PO-LIFT\0_K15\1\1SRef
0.0
0.5
1.0
4x10
Inte
ns. [
a.u.
]
632.401
1321.420179.584 794.323488.359 1545.392 1887.7661159.405
2009-11-10-BBG-PO-LIFT\0_K15\1\1887.0521.LIFT\1SRef
0.0
0.5
1.0
1.5
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
13Nature Methods: doi: 10.1038/nmeth.1540
Fraction 13 1613.941
1344.774 1823.095957.667
2009-11-10-BBG-PO-LIFT\0_K17\1\1SRef
0.0
0.5
1.0
1.5
4x10
Inte
ns. [
a.u.
]
632.604
956.488
1321.922
794.512 1614.7761159.607203.607 488.548
2009-11-10-BBG-PO-LIFT\0_K17\1\1613.9411.LIFT\1SRef
0.0
0.5
1.0
1.5
2.04x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 14 1614.195
1366.182 2024.360
2009-11-10-BBG-PO-LIFT\0_K19\1\1SRef
0
1000
2000
3000
4000
Inte
ns. [
a.u.
]
632.688
956.645 1322.234
1613.652369.719 1159.857
2009-11-10-BBG-PO-LIFT\0_K19\1\1614.1950.LIFT\1SRef
0.0
0.2
0.4
0.6
0.8
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 15 1099.571
1685.771
1412.668
2075.049
2009-11-10-BBG-PO-LIFT\0_K21\1\1SRef
0
500
1000
1500
Inte
ns. [
a.u.
]
1099.842
1662.064608.835 958.058335.831 1354.347
2009-11-10-BBG-PO-LIFT\0_K21\1\1685.7713.LIFT\1SRef
0
1000
2000
3000
4000
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
14Nature Methods: doi: 10.1038/nmeth.1540
Fraction 16 1817.4552009-11-10-BBG-PO-LIFT\0_K23\1\1SRef
0
500
1000
1500
Inte
ns. [
a.u.
]
633.522
204.128957.750
1527.667
366.115 1819.5911344.082
2009-11-10-BBG-PO-LIFT\0_K23\1\1817.4550.LIFT\1SRef
0
2000
4000
6000
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 17 1509.816
1218.654
2009-11-10-BBG-PO-LIFT\0_M1\1\1SRef
0
1000
2000
3000
Inte
ns. [
a.u.
]
1218.793
1509.916387.850 853.164153.965
2009-11-10-BBG-PO-LIFT\0_M1\1\1509.8162.LIFT\1SRef
0.0
0.5
1.0
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 18 713.371
1593.719
1302.5632031.096
2009-11-10-BBG-PO-LIFT\0_M3\1\1SRef
0
1000
2000
3000
Inte
ns. [
a.u.
]
1302.519
1593.796387.794 1099.145752.778
2009-11-10-BBG-PO-LIFT\0_M3\1\1593.7190.LIFT\1SRef
0.00
0.25
0.50
0.75
1.00
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
15Nature Methods: doi: 10.1038/nmeth.1540
Fraction 19 2020.099
1887.012
2229.279
2009-11-10-BBG-PO-LIFT\0_M5\1\1SRef
0
500
1000
1500
2000
2500
Inte
ns. [
a.u.
]
633.144
203.951 1730.095795.179365.812 1160.378 2021.801
2009-11-10-BBG-PO-LIFT\0_M5\1\2020.0990.LIFT\1SRef
0
2000
4000
6000
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 20 1522.028
957.770
1248.901795.663 1731.213
2009-11-10-BBG-PO-LIFT\0_M7\1\1SRef
0.0
0.5
1.0
4x10
Inte
ns. [
a.u.
]
633.238
1522.082957.378
795.313489.176187.989 1230.834
2009-11-10-BBG-PO-LIFT\0_M7\1\1522.0279.LIFT\1SRef
0
1000
2000
3000
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 21
1039.571 1782.876
1491.760 2084.257
2009-11-10-BBG-PO-LIFT\0_M9\1\1SRef
0
2000
4000
6000
Inte
ns. [
a.u.
]
1472.934
1200.570
657.785203.979 1720.1111038.321
2009-11-10-BBG-PO-LIFT\0_M9\1\1782.8760.LIFT\1SRef
0
2000
4000
6000
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
16Nature Methods: doi: 10.1038/nmeth.1540
Fraction 22 1874.979
1583.867 2078.026
2009-11-10-BBG-PO-LIFT\0_M11\1\1SRef
0
1000
2000
3000
Inte
ns. [
a.u.
]
603.194
1875.294
1585.119927.273 1323.853203.969
2009-11-10-BBG-PO-LIFT\0_M11\1\1874.9788.LIFT\1SRef
0
500
1000
1500
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 23 2108.300
2317.534
860.393
2009-11-10-BBG-PO-LIFT\0_M13\1\1SRef
0
1000
2000
3000
Inte
ns. [
a.u.
]
633.187
203.970
859.9681817.138
1248.395365.849 1555.423 2001.000
2009-11-10-BBG-PO-LIFT\0_M13\1\2108.3000.LIFT\1SRef
0.00
0.25
0.50
0.75
1.00
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 24 1905.150
1613.985795.593 1248.831 2114.317
2009-11-10-BBG-PO-LIFT\0_M15\1\1SRef
0.0
0.5
1.0
1.5
4x10
Inte
ns. [
a.u.
]
632.263
1593.459203.470 794.170 1322.319488.240 1905.069
2009-11-10-BBG-PO-LIFT\0_M15\1\1905.1498.LIFT\1SRef
0.0
0.2
0.4
0.6
0.8
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
17Nature Methods: doi: 10.1038/nmeth.1540
Fraction 25 1629.864
709.3451875.045 2210.192
1338.778
2009-11-10-BBG-PO-LIFT\0_M17\1\1SRef
0
500
1000
1500
2000
2500
Inte
ns. [
a.u.
]
656.920203.932
365.890
1338.831
973.279520.107
2009-11-10-BBG-PO-LIFT\0_M17\1\1629.8640.LIFT\1SRef
0
200
400
600
800
1000
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 26
1681.946
1390.798860.393
709.389
1099.637 2322.474
2009-11-10-BBG-PO-LIFT\0_M19\1\1SRef
0
2000
4000
6000
Inte
ns. [
a.u.
]
1370.8441098.417
414.536 733.303 1617.643185.587 980.748
2009-11-10-BBG-PO-LIFT\0_M19\1\1681.9460.LIFT\1SRef
0.0
0.5
1.0
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 27 2178.554
795.691 1905.3441614.180
2009-11-10-BBG-PO-LIFT\0_M21\1\1SRef
0.0
0.2
0.4
0.6
0.8
4x10
Inte
ns. [
a.u.
]
633.189
2179.864
795.226 1322.377 1888.4441613.877
2009-11-10-BBG-PO-LIFT\0_M21\1\2178.5538.LIFT\1SRef
0
250
500
750
1000
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
18Nature Methods: doi: 10.1038/nmeth.1540
Fraction 28 2178.255
1887.110
1651.906795.590 1360.792 2387.544
2009-11-10-BBG-PO-LIFT\0_M23\1\1SRef
0
2000
4000
6000
Inte
ns. [
a.u.
]
632.582
794.567 1321.478 1887.071203.650 1613.853488.464 2178.501
2009-11-10-BBG-PO-LIFT\0_M23\1\2178.2548.LIFT\1SRef
0.0
0.5
1.0
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 29 1887.030
1374.666 1677.7632178.135795.589 1113.564
2009-11-10-BBG-PO-LIFT\0_O1\1\1SRef
0
2000
4000
6000
8000
Inte
ns. [
a.u.
]
632.922
1322.159794.935365.651 1552.471 1887.554
2009-11-10-BBG-PO-LIFT\0_O1\1\1887.0300.LIFT\1SRef
0.00
0.25
0.50
0.75
1.004x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 30 1404.580
1113.463
1904.835
2009-11-10-BBG-PO-LIFT\0_O3\1\1SRef
0.00
0.25
0.50
0.75
1.004x10
Inte
ns. [
a.u.
]
1113.672
1404.708712.084388.039 550.042 959.304226.034
2009-11-10-BBG-PO-LIFT\0_O3\1\1404.5796.LIFT\1SRef
0
1
2
3
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
19Nature Methods: doi: 10.1038/nmeth.1540
Fraction 31
1801.090
1509.9391317.120
2009-11-10-BBG-PO-LIFT\0_O5\1\1SRef
0
2000
4000
6000
Inte
ns. [
a.u.
]
1490.4761218.125
852.837387.664 678.661 1737.5321100.329225.785
2009-11-10-BBG-PO-LIFT\0_O5\1\1801.0904.LIFT\1SRef
02000
40006000
8000
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 32 2193.972
713.3691920.872 2403.150
2009-11-10-BBG-PO-LIFT\0_O7\1\1SRef
0
2000
4000Inte
ns. [
a.u.
]
633.242
2195.3671322.491795.314 1614.146
2009-11-10-BBG-PO-LIFT\0_O7\1\2193.9719.LIFT\1SRef
0
200
400
600
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 33 2194.168
713.425
795.6111944.979
2009-11-10-BBG-PO-LIFT\0_O9\1\1SRef
0500
1000
15002000
Inte
ns. [
a.u.
]
633.125
795.230
2009-11-10-BBG-PO-LIFT\0_O9\1\2194.1683.LIFT\1SRef
0
200
400
600
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
20Nature Methods: doi: 10.1038/nmeth.1540
Fraction 34 2178.128
1905.005795.582 1613.9062387.307
2009-11-10-BBG-PO-LIFT\0_O11\1\1SRef
0.0
0.5
1.04x10
Inte
ns. [
a.u.
]
633.215
795.257 957.331 1322.442 1595.189204.010 1887.139
2009-11-10-BBG-PO-LIFT\0_O11\1\2178.1280.LIFT\1SRef
0
500
1000Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 35 2178.224
1887.056795.609 2387.3871613.968
2009-11-10-BBG-PO-LIFT\0_O13\1\1SRef
0.0
0.5
1.0
1.54x10
Inte
ns. [
a.u.
]
632.414
794.355 956.318 1321.230203.545 1885.729488.339 1613.396 2179.598
2009-11-10-BBG-PO-LIFT\0_O13\1\2178.2243.LIFT\1SRef
0.0
0.5
1.0
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 36 2177.943
757.399
845.470
889.484
1344.657
2009-11-10-BBG-PO-LIFT\0_O15\1\1SRef
0
1000
2000Inte
ns. [
a.u.
]
633.591
795.693 957.810 1323.041204.193 1889.0061596.770489.402 2179.113
2009-11-10-BBG-PO-LIFT\0_O15\1\2177.9000.LIFT\1SRef
0.0
0.5
1.0
1.5
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
21Nature Methods: doi: 10.1038/nmeth.1540
Fraction 37 2178.492
1614.145795.710 1905.3091248.955
2009-11-10-BBG-PO-LIFT\0_O17\1\1SRef
0.00
0.25
0.50
0.75
4x10
Inte
ns. [
a.u.
]
633.234
2181.005795.302 957.341 1322.392 1886.7251613.749546.832
2009-11-10-BBG-PO-LIFT\0_O17\1\2178.4916.LIFT\1SRef
0
500
1000
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 38 2451.852
2178.636
2091.6131887.429
2009-11-10-BBG-PO-LIFT\0_O19\1\1SRef
0
500
1000
1500
Inte
ns. [
a.u.
]
633.325
795.357546.947 957.347204.020 370.118 1323.344 1887.994 2453.0411553.937 2093.910
2009-11-10-BBG-PO-LIFT\0_O19\1\2451.8500.LIFT\1SRef
0
1000
2000
3000
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
Fraction 39
1157.4292178.427
1887.2762451.574
2009-11-19-BBG-PO-Redo\0_O21\1\1SRef
0
200
400
600
800
Inte
ns. [
a.u.
]
795.278370.166 1322.580 1887.9311613.947
2009-11-10-BBG-PO-LIFT\0_O21\1\2178.6120.LIFT\1SRef
0
50
100
150
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
22Nature Methods: doi: 10.1038/nmeth.1540
Fraction 40
1695.8541404.7361113.600
2009-11-10-BBG-PO-LIFT\0_O23\1\1SRef
0
2000
4000
6000
8000
Inte
ns. [
a.u.
]
1386.039
1113.714
984.936679.020387.961 1632.930
2009-11-10-BBG-PO-LIFT\0_O23\1\1695.8536.LIFT\1SRef
0.0
0.5
1.0
1.5
2.0
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500m/z
23Nature Methods: doi: 10.1038/nmeth.1540
Supplementary Table 2. Raw data of antibody binding from control and Lyme disease patient sera. (a) IgG response of sera from Lyme disease patients on the BBG-AOAB microarray. Average = average of 4 median RFU values.
PATIENT #1 PATIENT #2 PATIENT #3 PATIENT #4 PATIENT #5 PATIENT #6 PATIENT #7 PATIENT #8 PATIENT #9 PATIENT #10 Chart ID Average STDEV %CV Average STDEV %CV Average STDEV %CV Average STDEV %CV Average STDEV %CV Average STDEV %CV Average STDEV %CV Average STDEV %CV Average STDEV %CV Average STDEV %CV
1 1153 145 13 247 58 23 549 50 9 398 212 53 -152 498 -329 191 218 114 111 13 12 84 24 29 204 64 31 91 33 36
2 696 114 16 510 17 3 245 49 20 140 31 22 256 35 13 233 28 12 111 32 29 146 33 22 179 16 9 -7 94 -1340
3 1275 146 11 436 21 5 515 152 30 414 259 62 -117 474 -405 260 104 40 192 47 24 -113 154 -136 403 59 15 288 65 23
4 421 167 40 143 11 7 129 104 81 74 1 1 25 14 56 258 48 19 80 19 24 198 173 88 80 104 131 234 63 27
5 55 26 48 63 62 98 79 46 59 101 40 39 580 62 11 239 15 6 125 24 19 -31 60 -191 104 11 11 51 41 80
6 -8 29 -352 52 40 77 4 44 1108 29 3 10 247 5 2 223 79 35 140 68 49 69 19 28 -7 10 -150 41 31 76
7 727 197 27 230 42 18 118 32 27 487 183 38 342 245 72 196 93 48 48 16 34 80 109 136 -68 128 -188 32 40 127
8 358 45 13 62 8 14 73 18 25 7 7 100 68 51 75 290 113 39 19 10 53 -1 29 -5798 204 296 145 12 32 270
9 988 149 15 345 43 13 473 90 19 686 51 7 34 16 46 294 42 14 92 25 27 90 61 68 104 112 107 334 73 22
10 144 101 70 157 44 28 116 79 68 320 98 31 286 6 2 150 78 52 76 12 16 98 39 40 98 13 14 48 37 77
11 -31 58 -188 131 50 39 -5 273 -5198 175 40 23 -52 287 -551 136 47 34 63 6 10 83 73 88 64 170 266 14 6 40
12 7595 1809 24 1031 216 21 7242 1374 19 3170 325 10 97 202 207 626 92 15 140 38 27 690 65 9 -132 189 -143 2164 272 13
13 -33 299 -898 46 9 19 51 13 26 95 19 20 170 67 39 20 33 169 46 60 130 47 18 39 67 51 76 19 23 123
14 44 177 407 124 81 65 44 1 3 9 12 133 62 36 59 124 118 95 -52 7 -13 -32 60 -190 -38 168 -441 -30 54 -180
15 -40 41 -101 134 40 30 83 49 59 64 55 86 278 35 13 139 11 8 93 56 60 -6 52 -905 148 43 29 -16 154 -960
16 182 72 40 58 13 23 125 57 45 54 55 101 574 130 23 58 18 31 76 22 29 46 94 206 97 18 19 50 80 158
17 2238 296 13 774 35 5 609 127 21 574 233 41 109 44 41 130 94 73 373 88 24 369 69 19 -51 234 -459 476 23 5
18 -126 100 -80 357 79 22 237 111 47 238 8 4 320 152 48 89 11 13 158 42 26 194 26 14 55 52 95 213 20 9
19 -17 184 -1069 57 33 57 139 97 70 140 23 16 78 65 83 158 18 12 -18 94 -511 71 23 32 186 46 25 -52 29 -56
20 142 12 8 111 38 35 43 60 141 51 77 151 167 153 92 126 62 49 44 1 2 101 22 22 -137 183 -134 54 88 162
21 196 46 23 98 16 16 8 15 182 -4 9 -223 129 74 57 68 70 104 137 48 35 93 34 36 -46 63 -137 32 14 44
22 114 51 45 61 12 19 -29 56 -195 54 24 45 461 106 23 -29 100 -343 36 15 42 -64 55 -87 39 11 29 -69 55 -80
23 40 138 349 227 55 24 38 31 80 128 2 2 -131 238 -182 57 107 189 -55 52 -95 -103 142 -138 188 107 57 47 63 135
24 364 424 116 218 14 6 -16 86 -523 60 90 152 487 315 65 241 33 14 155 36 23 123 18 15 -3 53 -1630 75 18 24
25 266 53 20 186 42 22 43 28 65 160 47 30 -4 48 -1197 272 44 16 40 58 144 -20 41 -205 127 33 26 -66 97 -148
26 167 285 170 685 226 33 1839 395 21 1017 209 21 752 36 5 591 165 28 313 30 9 770 269 35 98 267 274 861 180 21
27 -36 49 -136 237 67 28 261 59 23 177 12 7 312 118 38 179 112 63 17 32 183 -23 8 -36 193 148 77 52 34 65
28 115 45 39 253 71 28 116 28 24 49 12 25 -132 735 -557 334 169 51 19 45 238 353 27 8 90 45 50 17 16 92
29 304 33 11 146 48 33 237 67 28 6 10 150 331 73 22 44 70 158 10 37 368 -38 22 -59 32 69 217 -31 58 -191
30 132 34 25 214 23 11 97 16 16 24 3 13 1163 179 15 103 13 12 -28 51 -182 124 30 24 238 112 47 92 7 7
31 -8 104 -1304 113 29 26 61 40 66 31 38 122 -75 474 -630 138 20 14 34 30 88 -70 64 -92 77 22 29 53 43 82
32 214 68 32 69 4 6 91 26 29 11 4 34 221 112 51 134 88 65 -15 60 -391 49 79 162 -4 197 -4927 8 14 168
33 2781 146 5 1276 336 26 2539 587 23 1232 302 25 824 199 24 435 105 24 722 33 5 1157 172 15 807 127 16 1020 222 22
34 228 34 15 230 42 18 39 101 263 111 28 25 15 53 366 55 60 108 91 33 36 50 9 19 -6 146 -2546 77 31 41
35 250 91 37 325 50 15 -21 28 -136 246 94 38 32 339 1072 323 81 25 99 16 16 169 48 28 274 78 29 195 34 18
36 557 62 11 126 109 86 147 40 28 85 57 68 238 316 133 162 183 113 32 38 119 185 44 24 96 70 73 63 56 89
37 -50 124 -249 4 9 202 83 20 25 23 25 108 400 175 44 291 145 50 -3 37 -1242 -1 11 -2132 -63 74 -117 -19 58 -300
38 -3 79 -2637 32 16 52 -36 34 -96 26 40 152 -43 435 -1024 234 84 36 67 29 43 -46 99 -215 41 12 30 21 111 539
39 469 139 30 728 205 28 148 28 19 560 120 21 495 100 20 429 203 47 126 35 28 279 31 11 195 49 25 635 169 27
40 529 125 24 341 257 75 463 109 24 112 144 129 2681 940 35 698 311 45 205 48 23 470 116 25 585 103 18 698 124 18
41 991 164 17 206 37 18 160 33 21 193 81 42 -220 324 -148 188 43 23 11 4 39 195 57 29 -71 61 -85 59 12 21
42 1001 337 34 138 40 29 143 16 11 145 59 41 525 336 64 209 41 19 0 24 -7202 285 59 21 54 25 46 44 15 33
43 131 33 25 50 11 21 43 74 171 46 12 27 1465 234 16 0 11 -3208 67 24 36 -128 84 -66 -96 145 -152 2 25 1050
44 863 129 15 326 66 20 162 26 16 93 50 54 714 204 29 160 91 57 197 24 12 305 75 25 245 100 41 423 105 25
Nature Methods: doi: 10.1038/nmeth.1540
CONTROL #1 CONTROL #2 CONTROL #3 CONTROL #4 CONTROL #5 CONTROL #6 CONTROL #7 CONTROL #8 Chart ID Average STDEV %CV Average STDEV %CV Average STDEV %CV Average STDEV %CV Average STDEV %CV Average STDEV %CV Average STDEV %CV Average STDEV %CV
1 373 257 69 -5 34 -716 -152 31 -355 191 3 22 111 17 59 84 8 42 21 4 20 49 6 13 2 322 71 22 92 62 67 256 48 35 233 2 9 111 10 12 146 4 16 19 6 30 47 8 17 3 -151 435 -289 17 6 36 -117 35 29 260 5 15 192 10 11 -113 9 29 37 7 17 179 42 23 4 149 221 148 7 11 162 25 17 25 258 13 85 80 19 43 198 6 35 22 9 41 84 15 18 5 0 29 -8779 43 16 36 580 20 29 239 1 11 125 15 -196 -31 9 36 13 3 20 44 12 27 6 12 14 118 19 2 12 247 35 462 223 3 24 140 24 -4760 69 3 21 17 5 31 26 11 42 7 303 87 29 19 1 5 342 12 12 196 5 26 48 16 31 80 3 14 14 1 7 48 38 79 8 284 105 37 11 16 141 68 244 -2122 290 5 21 19 25 3703 -1 3 21 11 1 13 50 18 36 9 676 291 43 7 12 167 34 4 5 294 4 20 92 9 15 90 13 34 21 5 22 68 5 8
10 190 115 61 32 7 22 286 46 42 150 4 27 76 7 18 98 0 0 12 5 41 47 11 23 11 -42 122 -295 20 4 18 -52 102 -1388 136 2 10 63 7 13 83 7 22 17 2 14 56 11 20 12 -67 134 -202 61 7 11 97 40 32 626 9 30 140 36 22 690 17 24 41 10 23 111 50 45 13 -15 21 -140 61 5 9 170 30 31 20 4 28 46 0 0 47 10 31 13 0 0 24 9 39 14 53 38 72 41 42 103 62 5 9 124 3 22 -52 7 39 -32 1 4 14 1 10 20 4 18 15 -16 85 -522 21 14 67 278 67 -256 139 10 55 93 8 35 -6 4 25 12 1 12 35 1 4 16 -8 68 -821 54 30 56 574 37 -64 58 2 21 76 15 72 46 13 78 11 2 17 16 8 49 17 866 169 20 66 18 27 109 13 12 130 9 26 373 22 18 369 7 12 39 7 18 106 19 17 18 286 87 30 47 20 43 320 56 35 89 7 42 158 12 18 194 8 27 20 1 6 59 11 18 19 54 76 141 27 2 8 78 20 50 158 4 17 -18 7 47 71 7 29 19 2 8 50 2 3 20 237 62 26 29 31 106 167 22 43 126 5 30 44 6 21 101 3 11 14 4 26 33 4 13 21 21 5 25 29 15 51 129 61 -574 68 4 27 137 10 -104 93 4 25 17 3 20 24 1 4 22 17 4 20 5 14 304 461 28 -704 -29 3 22 36 8 87 -64 13 53 15 4 28 22 1 3 23 109 92 84 46 14 30 -131 1 1 57 3 27 -55 5 14 -103 4 28 14 6 47 18 12 66 24 312 319 102 13 13 99 487 94 -568 241 3 29 155 40 -269 123 3 9 14 3 18 33 7 22 25 134 32 24 7 5 76 -4 4 21 272 2 23 40 16 77 -20 5 15 17 7 43 11 3 24 26 405 180 44 121 1 1 752 289 25 591 21 35 313 57 66 770 12 12 117 26 23 104 10 10 27 937 86 9 29 17 59 312 79 88 179 5 26 17 9 25 -23 9 53 22 4 18 50 10 20 28 332 115 35 25 13 54 -132 38 34 334 5 28 19 13 42 353 8 20 22 4 21 23 8 35 29 31 12 38 10 19 191 331 32 41 44 8 55 10 17 77 -38 3 11 11 1 13 23 0 0 30 -456 284 -62 7 5 71 1163 25 20 103 8 38 -28 12 21 124 7 17 15 2 15 42 7 17 31 -138 146 -106 1 12 889 -75 43 -445 138 4 19 34 20 50 -70 9 49 21 4 20 16 3 18 32 218 39 18 13 14 108 221 2 2 134 7 31 -15 18 87 49 1 9 16 4 23 33 9 26 33 1866 251 13 221 37 17 824 45 15 435 49 24 722 85 25 1157 2 1 159 33 21 241 60 25 34 55 103 188 27 10 37 15 63 26 55 3 13 91 30 160 50 7 23 24 5 21 52 13 25 35 -95 128 -135 9 28 310 32 11 5 323 6 17 99 23 33 169 3 17 22 4 20 29 9 32 36 -18 221 -1225 29 29 97 238 12 9 162 5 37 32 31 38 185 5 12 22 3 11 19 17 89 37 -21 52 -243 28 4 12 400 116 157 291 2 9 -3 17 50 -1 8 41 18 2 12 8 13 157 38 108 38 35 60 2 3 -43 233 129 234 7 15 67 6 17 -46 18 129 50 11 23 26 28 106 39 753 331 44 38 33 87 495 195 38 429 27 66 126 9 10 279 3 7 51 2 4 59 11 19 40 99 91 92 144 25 17 2681 173 57 698 12 24 205 12 8 470 25 19 57 5 9 89 18 20 41 -68 95 -140 76 30 39 -220 60 16 188 16 47 11 7 18 195 11 28 21 2 9 41 7 17 42 -64 146 -228 56 33 59 525 44 8 209 6 15 0 4 6 285 16 -466 38 11 29 36 10 28 43 -360 303 -84 13 11 87 1465 44 69 0 11 84 67 43 202 -128 6 182 14 3 20 16 6 35 44 224 166 74 66 17 25 714 93 17 160 0 0 197 60 38 305 49 113 37 12 32 93 23 25
(b) IgG response of control sera on the BBG-AOAB microarray. Average = average of 4 median RFU values.
Nature Methods: doi: 10.1038/nmeth.1540
Supplementary Table 3. MALDI-TOF MS and MS/MS data of the 10 peaks collected from C18 HPLC separation of PC3 cell GSL-AOABs. For each fraction, MS at positive mode, MS at negative mode and MS/MS at positive mode were obtained and shown below. Fraction 3
1494.645
964.418
1153.540
1793.734
843.369
821.391 1348.590
2062.877
2010-06-15-PC3 33 fractions \0_B5\1\1SRef
0
500
1000
1500Inte
ns. [
a.u.
]
1550.944
1009.679
1748.1241390.887
1160.702
2010-06-15-PC3 33 fractions \0_B5\2\1SRef
0
100
200
300
Inte
ns. [
a.u.
]
967.155
357.8921106.767
621.566
1309.331
783.1891479.427
2010-06-15-PC3 33 fractions \0_B5\2\1494.6000.LIFT\1SRef
0
100
200
300
400
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500 3000m/z
Nature Methods: doi: 10.1038/nmeth.1540
Fraction 5
1151.822
852.715
1995.319
1720.148
1338.968993.615
1438.914 2140.386
2010-06-15-PC3 33 fractions \0_B9\1\1SRef
0.0
0.5
1.0
1.5
4x10
Inte
ns. [
a.u.
]
1993.520
1023.766
1798.3521221.003
1361.0072284.663
1630.209
2010-06-15-PC3 33 fractions \0_B9\2\1SRef
0
250
500
750
1000
1250
1500
Inte
ns. [
a.u.
]
852.758
1339.168
1705.915
1014.964 1542.504204.147 589.489
2010-06-15-PC3 33 fractions \0_B9\2\1995.3000.LIFT\1SRef
0
500
1000
1500
2000
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500 3000m/z
Nature Methods: doi: 10.1038/nmeth.1540
Fraction 6
852.645
1704.0671165.745
1483.938
1849.132
1338.8722124.303
1875.066
2010-06-15-PC3 33 fractions \0_B11\1\1SRef
0
2000
4000
6000
8000
Inte
ns. [
a.u.
]
1782.243
1636.174
1009.7131482.145
1163.936 1963.393
2010-06-15-PC3 33 fractions \0_B11\2\1SRef
0
500
1000
1500
Inte
ns. [
a.u.
]
852.368
1395.159
1176.614203.928
978.431
589.155 1542.253
1215.091
2010-06-15-PC3 33 fractions \0_B11\2\1704.1000.LIFT\1SRef
0
500
1000
1500
2000
2500
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500 3000m/z
Nature Methods: doi: 10.1038/nmeth.1540
Fraction 8
1438.790
1218.690
934.554 1583.8611073.629
1858.975
1615.882
2010-06-15-PC3 33 fractions \0_B15\1\1SRef
0
2000
4000
6000
Inte
ns. [
a.u.
]
1517.005
1370.922
1216.908
1762.2201005.666
1662.053
2010-06-15-PC3 33 fractions \0_B15\2\1SRef
0
1000
2000
3000
4000
5000
Inte
ns. [
a.u.
]
603.362
1130.396
204.110
911.737
1297.820366.139
2010-06-15-PC3 33 fractions \0_B15\2\1438.8000.LIFT\1SRef
0
200
400
600
800
1000
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500 3000m/z
Nature Methods: doi: 10.1038/nmeth.1540
Fraction 9
1202.709
927.580 1405.788
1567.850
2010-06-15-PC3 33 fractions \0_B17\1\1SRef
0.0
0.2
0.4
0.6
0.8
1.0
1.24x10
Inte
ns. [
a.u.
]
1005.662
1354.918
1200.905
1566.079
1516.973
2153.4871860.340
2010-06-15-PC3 33 fractions \0_B17\2\1SRef
0
200
400
600
800
1000
Inte
ns. [
a.u.
]
911.343
587.023
749.1601192.747359.868
2010-06-15-PC3 33 fractions \0_B17\2\1202.7000.LIFT\1SRef
0
100
200
300
400
500
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500 3000m/z
Nature Methods: doi: 10.1038/nmeth.1540
Fraction 15
1748.007
1247.593
929.6311499.501 1893.026
1909.004
2010-06-15-PC3 33 fractions \0_D5\1\1SRef
0.0
0.5
1.0
1.5
4x10
Inte
ns. [
a.u.
]
1746.236
1303.835
1141.750
1551.065
1579.0801440.988
1882.2641004.586
2314.554
2010-06-15-PC3 33 fractions \0_D5\2\1SRef
0
1000
2000
3000
4000
Inte
ns. [
a.u.
]
604.353
1090.308
1455.558
928.2741293.385203.469 460.292 1747.208
2010-06-15-PC3 33 fractions \0_D5\2\1748.0000.LIFT\1SRef
0
2000
4000
6000Inte
ns. [
a.u.
]
500 1000 1500 2000 2500 3000m/z
Nature Methods: doi: 10.1038/nmeth.1540
Fraction 16
1456.820
1091.663
1601.847
2047.009808.313
1747.892
2010-06-15-PC3 33 fractions \0_D7\1\1SRef
0.0
0.5
1.0
1.5
2.0
4x10
Inte
ns. [
a.u.
]
1535.086
1389.002
2010-06-15-PC3 33 fractions \0_D7\2\1SRef
0
500
1000
1500
2000
Inte
ns. [
a.u.
]
604.673
1090.805
928.664341.711 1456.205766.598
2010-06-15-PC3 33 fractions \0_D7\2\1456.8000.LIFT\1SRef
0.00
0.25
0.50
0.75
1.00
1.25
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500 3000m/z
Nature Methods: doi: 10.1038/nmeth.1540
Fraction 18
1236.755
1585.900
1011.514
1423.838
1788.975
2010-06-15-PC3 33 fractions \0_D13\1\1SRef
0
1000
2000
3000
4000
5000
Inte
ns. [
a.u.
]
1584.222
1235.013
1373.047
1009.690 1787.350
1367.993
2010-06-15-PC3 33 fractions \0_D13\2\1SRef
0
500
1000
1500
2000
2500
Inte
ns. [
a.u.
]
604.449
1293.949
766.338203.493 1131.507460.401 1585.425
2010-06-15-PC3 33 fractions \0_D13\2\1585.9000.LIFT\1SRef
0.00
0.25
0.50
0.75
1.00
1.25
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500 3000m/z
Nature Methods: doi: 10.1038/nmeth.1540
Fraction 20
1220.848
974.244
1556.028 1847.183
2010-06-15-PC3 33 fractions \0_D17\1\1SRef
0.0
0.5
1.0
1.5
2.0
2.5
4x10
Inte
ns. [
a.u.
]
1219.071
1023.845
1373.112
1529.185
2010-06-15-PC3 33 fractions \0_D17\2\1SRef
0
500
1000
1500
Inte
ns. [
a.u.
]
604.313
928.520
1195.982341.380 766.335
2010-06-15-PC3 33 fractions \0_D17\2\1220.8000.LIFT\1SRef
0.00
0.25
0.50
0.75
1.00
1.25
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500 3000m/z
Nature Methods: doi: 10.1038/nmeth.1540
Fraction 21
929.707
1190.831
1393.923
2010-06-15-PC3 33 fractions \0_D19\1\1SRef
0.0
0.5
1.0
1.5
2.0
4x10
Inte
ns. [
a.u.
]
1007.771
1317.9151155.827
1593.163
2010-06-15-PC3 33 fractions \0_D19\2\1SRef
0.0
0.2
0.4
0.6
0.8
1.0
4x10
Inte
ns. [
a.u.
]
605.791
342.140921.108
2010-06-15-PC3 33 fractions \0_D19\2\929.7000.LIFT\1SRef
0
500
1000
1500
2000
2500
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500 3000m/z
Nature Methods: doi: 10.1038/nmeth.1540