shotgun glycomics: a microarray strategy for functional ... · nature methods shotgun glycomics: a...

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.


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).


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


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.


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


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.


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.


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.


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


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


(b) Re-chromatography of all 40 HPLC fractions on C18 HPLC. F-01

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

-0.0025

0.0000

0.0025

0.0050

0.0075

0.0100

0.0125

0.0150

0.0175

0.0200

0.0225

0.0250RF-10AxlCalbiochem-BBG-PO-NP01-RP01

F-02

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.010 RF-10AxlCalbiochem-BBG-PO-NP02-RP03

F-03

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.010

RF-10AxlCalbiochem-BBG-PO-NP03-RP02

F-04

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.00

0.02

0.04

0.06

0.08

0.10


F-05

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040



F-06

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08


F-07

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.000

0.005

0.010

0.015

0.020

0.025


F-08

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18


F-09

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45


F-10

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

-0.0025

0.0000

0.0025

0.0050

0.0075

0.0100

0.0125

0.0150

0.0175

0.0200



F-11

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14


F-12

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.00

0.05

0.10

0.15

0.20

0.25

0.30


F-13

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14


F-14

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.000

0.005

0.010

0.015

0.020

0.025

0.030


F-15

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.00

0.02

0.04

0.06

0.08

0.10

0.12



F-16

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

-0.0025

0.0000

0.0025

0.0050

0.0075

0.0100

0.0125

0.0150

0.0175

0.0200

0.0225


F-17

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

-0.0025

0.0000

0.0025

0.0050

0.0075

0.0100

0.0125

0.0150

0.0175

0.0200

0.0225


F-18

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.000

0.001

0.002

0.003

0.004

0.005


F-19

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.000

0.002

0.004

0.006

0.008

0.010


F-20

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.00

0.01

0.02

0.03

0.04

0.05



F-21

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

-0.0025

0.0000

0.0025

0.0050

0.0075

0.0100

0.0125

0.0150

0.0175

0.0200

0.0225


F-22

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16


F-23

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16


F-24

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7


F-25

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035



F-26

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16


F-27, 28, 29 (left, middle and right peaks respectively)

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.00

0.05

0.10

0.15

0.20

0.25


F-30

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.00

0.01

0.02

0.03

0.04

0.05

0.06


F-31

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.000

0.005

0.010

0.015

0.020

0.025

0.030


F-32, 33 (left and right peaks respectively)

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014




Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.00

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0.35



Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

-0.01

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09



Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016


F-40

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Volts

0.000

0.005

0.010

0.015

0.020

0.025

0.030



(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


0

2000

4000

6000

8000

Inte

ns. [

a.u.

]

633.770

1320.783

957.113370.087795.465 1159.549488.961


0

2000

4000

6000

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


1823.134

1298.674


0

1000

2000

3000

4000

Inte

ns. [

a.u.

]

632.733

1322.181 1614.722794.638365.532


0

1000

2000

3000

4000

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z



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


0

1

2

3

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


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


0.0

0.5

1.0

1.5

4x10

Inte

ns. [

a.u.

]

603.079

1292.585

765.050365.791 1585.2511157.977


0

1

2

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z



1614.061

2096.376


0

2000

4000

6000

Inte

ns. [

a.u.

]

632.627

1596.831956.687179.703 435.387 1885.6301248.842


0.0

0.5

1.0

1.5

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


1085.5321886.987


0

1000

2000

Inte

ns. [

a.u.

]

1069.071

1359.312387.956 704.184 951.204


0.0

0.5

1.0

1.5

2.04x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


1365.964 1823.219795.610


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


0.0

0.5

1.0

1.5

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z



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


0

500

1000

1500

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


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


0.0

0.5

1.0

1.5

2.0

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


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


0.0

0.5

1.0

1.5

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z



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


0.0

0.5

1.0

1.5

2.04x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


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


0.0

0.2

0.4

0.6

0.8

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


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


0

1000

2000

3000

4000

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


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


0

2000

4000

6000

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


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


0

1000

2000

3000

Inte

ns. [

a.u.

]

1302.519

1593.796387.794 1099.145752.778


0.00

0.25

0.50

0.75

1.00

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z



1887.012

2229.279


0

500

1000

1500

2000

2500

Inte

ns. [

a.u.

]

633.144

203.951 1730.095795.179365.812 1160.378 2021.801


0

2000

4000

6000

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


957.770

1248.901795.663 1731.213


0.0

0.5

1.0

4x10

Inte

ns. [

a.u.

]

633.238

1522.082957.378

795.313489.176187.989 1230.834


0

1000

2000

3000

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z

Fraction 21

1039.571 1782.876

1491.760 2084.257


0

2000

4000

6000

Inte

ns. [

a.u.

]

1472.934

1200.570

657.785203.979 1720.1111038.321


0

2000

4000

6000

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z



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


0

500

1000

1500

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


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


0.00

0.25

0.50

0.75

1.00

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


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


0.0

0.2

0.4

0.6

0.8

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z



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


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


0.0

0.5

1.0

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


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


0

250

500

750

1000

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z



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


0.0

0.5

1.0

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


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


1113.463

1904.835


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


0

1

2

3

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


Fraction 31

1801.090

1509.9391317.120


0

2000

4000

6000

Inte

ns. [

a.u.

]

1490.4761218.125

852.837387.664 678.661 1737.5321100.329225.785


02000

40006000

8000

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


713.3691920.872 2403.150


0

2000

4000Inte

ns. [

a.u.

]

633.242

2195.3671322.491795.314 1614.146


0

200

400

600

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


713.425

795.6111944.979


0500

1000

15002000

Inte

ns. [

a.u.

]

633.125

795.230


0

200

400

600

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z



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


0

500

1000Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


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


0.0

0.5

1.0

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


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


0.0

0.5

1.0

1.5

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z



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


0

500

1000

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


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


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


0

50

100

150

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


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


0.0

0.5

1.0

1.5

2.0

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z


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


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.


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


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


Fraction 5

1151.822

852.715

1995.319

1720.148

1338.968993.615

1438.914 2140.386


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


0

250

500

750

1000

1250

1500

Inte

ns. [

a.u.

]

852.758

1339.168

1705.915

1014.964 1542.504204.147 589.489


0

500

1000

1500

2000

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500 3000m/z


Fraction 6

852.645

1704.0671165.745

1483.938

1849.132

1338.8722124.303

1875.066


0

2000

4000

6000

8000

Inte

ns. [

a.u.

]

1782.243

1636.174

1009.7131482.145

1163.936 1963.393


0

500

1000

1500

Inte

ns. [

a.u.

]

852.368

1395.159

1176.614203.928

978.431

589.155 1542.253

1215.091


0

500

1000

1500

2000

2500

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500 3000m/z


Fraction 8

1438.790

1218.690

934.554 1583.8611073.629

1858.975

1615.882


0

2000

4000

6000

Inte

ns. [

a.u.

]

1517.005

1370.922

1216.908

1762.2201005.666

1662.053


0

1000

2000

3000

4000

5000

Inte

ns. [

a.u.

]

603.362

1130.396

204.110

911.737

1297.820366.139


0

200

400

600

800

1000

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500 3000m/z


Fraction 9

1202.709

927.580 1405.788

1567.850


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


0

200

400

600

800

1000

Inte

ns. [

a.u.

]

911.343

587.023

749.1601192.747359.868


0

100

200

300

400

500

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500 3000m/z


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


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


Fraction 16

1456.820

1091.663

1601.847

2047.009808.313

1747.892


0.0

0.5

1.0

1.5

2.0

4x10

Inte

ns. [

a.u.

]

1535.086

1389.002


0

500

1000

1500

2000

Inte

ns. [

a.u.

]

604.673

1090.805

928.664341.711 1456.205766.598


0.00

0.25

0.50

0.75

1.00

1.25

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500 3000m/z


Fraction 18

1236.755

1585.900

1011.514

1423.838

1788.975


0

1000

2000

3000

4000

5000

Inte

ns. [

a.u.

]

1584.222

1235.013

1373.047

1009.690 1787.350

1367.993


0

500

1000

1500

2000

2500

Inte

ns. [

a.u.

]

604.449

1293.949

766.338203.493 1131.507460.401 1585.425


0.00

0.25

0.50

0.75

1.00

1.25

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500 3000m/z


Fraction 20

1220.848

974.244

1556.028 1847.183


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


0

500

1000

1500

Inte

ns. [

a.u.

]

604.313

928.520

1195.982341.380 766.335


0.00

0.25

0.50

0.75

1.00

1.25

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500 3000m/z


Fraction 21

929.707

1190.831

1393.923


0.0

0.5

1.0

1.5

2.0

4x10

Inte

ns. [

a.u.

]

1007.771

1317.9151155.827

1593.163


0.0

0.2

0.4

0.6

0.8

1.0

4x10

Inte

ns. [

a.u.

]

605.791

342.140921.108


0

500

1000

1500

2000

2500

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500 3000m/z


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