John A. Goetz 1,2 , Michael G. House 3 , Milos V. Novotny 1,2,3

1. 2X Monitoring Changes in Serum Glycosylation Following Resection of Intraductal Papillary MucinousNeoplasms 6 24 5 100 2.1E+4 20 1850.1 90 100 100 979.7 1.7E+4 80 90 90 4 2808.4 70 16 80 80 2808.4 60 70 70 3 50 60 60 12 John A. Goetz1,2, Michael G. House3 ,MilosV. Novotny1,2,3 1Department of Chemistry, Indiana University, Bloomington, Indiana, 2National Center for Glycomics and Glycoproteomics, Bloomington, Indiana, 3Indiana University, School of Medicine, Indianapolis, Indiana 40 50 50 30 2447.3 40 2 40 2447.2 2056.7 20 30 8 1384.6 1852.4 2982.5 3618.8 30 490.2 3792.9 2056.1 1852.0 2621.4 1592.8 1799.9 2260.2 3257.7 2866.4 1595.8 2301.2 2982.4 2896.4 10 2412.3 3227.7 20 4604.1 4069.0 4243.1 4430.1 2621.3 4778.2 1923.0 2004.0 2260.0 2208.1 3431.8 3966.0 2288.0 3618.9 2866.4 1595.8 3792.7 1 2097.8 20 1800.3 2412.1 2505.3 3257.3 3227.3 2896.4 2003.8 2207.9 1882.5 10 0 3431.2 1677.3 4428.2 4241.7 4603.6 4068.2 260.1 1500 2200 2900 3600 4300 5000 1125.5 10 4 0 1500 2200 2900 3600 4300 5000 0 0 49.0 430.6 812.2 1193.8 1575.4 1957.0 0 Principal Component Analysis of MALDI Mass Spectra Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Introduction 1595.8 1799.9 2004.0 2208.1 2412.2 1851.9 2056.0 2260.1 2621.3 2982.5 MALDI TOF/TOF Spectra of Selected Ions Pre- and Post-Resection Glycosylation of proteins is a prevailing post-translational modification which governs folding, stability and function of the modified proteins. Aberrant glycosylation has been linked to malignant transformation for decades and quantitative glycomic measurements could play a significant role in the detection of adenocarcinomas of the lung, breast and colon. In this study, we examined the asparagine-linked (N-linked) oligosaccharides found in human blood serum before and after surgical resection of intraductal papillary mucinousneoplasms (IPMN). IPMN is of interest since it is considered an early stage of pancreatic cancer as the seemingly benign tumors have the ability to become invasive over time. IPMN is the subject of controversy since the broad spectrum of this disease results in difficulty in accurately diagnosing the tumors and vastly different recommendations for treatment. In this study, we have used human blood serum obtained from patients before and after surgical resection to monitor the levels of N-linked oligosaccharides. The samples were collected within five days of surgery, and we were able to identify multiple N-linked oligosaccharides which changed significantly in that relatively short time-frame. Principal Component Analysis (PCA) was performed using the spectra resulting from the MALDI-MS analysis of the serum N-glycans. PCA is a method that depicts the data graphically based on the variability of the samples. The PCA graph was made using MiniTab statistical analysis software and was calculated based on a two-factor analysis of covariance. As seen below, the samples pre- and post-resection cluster together indicating similarities between the two groups. We examined the possibility that the increases in relative intensity of selected N-glycans was the result of different compositions of saccharides or isomers of the structures. For this investigation we performed MALDI TOF/TOF analysis of the samples and fragmented the selected ions. Fragments were then matched and structures were verified using GlycoWorkbench software (www.eurocarbdb.org/applications/ms-tools). 2 5 1 5 0 S b 1 0 0 S b 9 3 S b Pre-Resection (n=34) 1 1 2 S 2 0 Post-Resection (n=23) 4 3 1 S 1 5 t 1 0 0 S n 9 3 S 1 1 9 S e n 5 4 9 S b 1 0 o 1 0 8 S b 2 2 8 1 5 S 2 b S b p 4 3 1 S b 1 9 4 S b 2 3 3 S b m 2 9 S b 3 5 S 4 7 1 S b 2 2 8 S 3 9 6 S b o 9 1 S b 5 5 1 S b 2 3 1 S b 2 1 9 S 3 7 8 S b 3 9 S b 2 3 3 S C 5 1 0 9 S b 1 0 8 S 1 5 2 S 5 5 1 S 3 3 S 5 4 9 S 2 2 S d 1 6 9 S b 3 5 5 S 2 9 S 3 9 6 S n 9 5 S b 3 9 S 2 3 1 S 2 5 S o 2 2 S b 2 6 3 S 3 3 S b 4 7 S 1 2 S c 0 1 5 0 S 4 7 1 S Sample Preparation Flow Diagram e 9 1 S S 9 5 S 1 9 4 S 3 7 8 S 2 2 1 S - 5 2 2 3 S 1 6 9 S Begin with 2.5ml of blood serum - 1 0 1 9 0 S Denature proteins using SDS and b-mercaptoethanol MALDI-TOF/TOF Tandem Mass Spectra of molecular ion at m/z 1852 from Patient 549 Pre-Resection - 6 0 - 5 0 - 4 0 - 3 0 - 2 0 - 1 0 0 F i r s t C o m p o n e n t Box Plots Depicting the Individual Values of N-glycan Relative Intensities from MALDI Mass Spectra Digest sample using PNGase F at 37°C for 24 hours Clean up samples using activated charcoal for solid phase extraction Box Plots were made using Origin 8.1 software depicting the individual values of the relative intensities for individual N-glycans from the MALDI mass spectra. These plots demonstrate changes in the composition of N-glycans in the spectra in response to surgical resection of the IPMN. The tables below are broken into different groups of glycans that exhibit significant changes. 1850.1 100 312.1 90 Dry samples 1384.6 80 1592.8 490.2 70 60 Resuspend in permethylation mixture 50 40 260.1 30 Perform spin-column permethylation 20 1125.2 10 0 49.0 430.6 812.2 1193.8 1575.4 1957 Perform liquid-liquid extraction on samples MALDI-TOF/TOF Tandem Mass Spectra of molecular ion at m/z 1852 from Patient 549 Post-Resection Dry samples and resuspend for analysis Analyze samples using MALDI-MS 2056.0 100 411.6 Materials and Methods 90 80 70 1588.7 We have generated N-glycanglycomic maps from patients before and after surgical resection of intraductal papillary ductalneoplasms. 2.5 ml of human blood serum was denatured using a combination of heat, SDS and b-mercaptoethanol in preparation for enzymatic digestion. PNGase F was then added to each samples and digestion was performed for 24 hours at 37° C. Samples were then subjected to solid-phase extraction using activated graphite columns to purify the free N-glycans. Samples were subsequently dried and then a solid-phase permethylation was performed using the spin-column technique1. Samples were first dissolved in 70ml of DMF, 5ml of water and 25ml of iodomethane and then incubated on a spin-column packed with sodium hydroxide mesh beads. After 15 minutes the samples were centrifuged, an additional 25ml of iodomethane was added to each sample and they were incubated for a second time on the sodium hydroxide column. Liquid-liquid extraction was then performed and samples were dried. The residues were resolubilized using 10ml of a 50:50 water/methanol mixture and analyzed using MALDI-MS. N-glycans of interest were then subjected to analysis by MS/MS to determine composition and isomeric information. 60 490.2 1592.8 50 1796.9 40 30 20 1595.6 1125.4 10 0 The above graph indicates changes in biantennary N-glycans which are fucosylated, changing significantly from pre- and post-resection samples. We see significant increases in the first three members of the group and we actually observe a slight decrease in the final member. 49.0 473.6 898.2 1322.8 1747.4 2172.0 MALDI-TOF/TOF Tandem Mass Spectra of molecular ion at m/z 2056 from Patient 549 Pre-Resection 2054.0 100 433.1 90 80 70 1608.7 60 1588.8 50 40 1592.8 490.2 30 1797.8 2040.1 MALDI-TOF Mass Spectra of N-glycans Released from Blood Serum of Patient 549 Pre-Resection 20 1333.6 2002.0 1125.2 10 0 49.0 473.6 898.2 1322.8 1747.4 2172.0 MALDI-TOF/TOF Tandem Mass Spectra of molecular ion at m/z 2056 from Patient 549 Post-Resection MALDI-TOF mass spectra were acquired for each sample using a Applied Biosystems 4800 MALDI TOF/TOF Analyzer. Below is a representative spectrum demonstrating typical pre-resection results from patient 549 Conclusions We performed glycomic analysis of N-linked glycans in the blood serum of patients pre- and post-resection of IPMN. Out of 54 glycans investigated, we found significant changes among 14 structures. We found significant changes in two groups of N-glycans: high-mannose and complex biantennary glycans with fucose. The changes in the high-mannose group were consistent across the profile of five structures. The changes that were most evident belong to a group of complex biantennary glycans, which are fucosylated. This is consistent with previous findings that there is an increase in N-linked oligosaccharide fucosylation associated with pancreatic tumors. Furthermore, fragmentation of these glycans reveals that we are not seeing any additional isomers, indicating the increases we observe can be attributed to one glycan. These findings indicate changes in the N-linked oligosaccharides of human blood serum, which, are most likely attributable to altered glycosylation of immunoglobulins. This is based on the fact that immunoglobulins are known to be modified extensively by all of the glycans which we have observed as significantly altered. The above graph indicates changes in high-mannose N-glycans which change significantly from pre- and post-resection samples. We see significant increases in the first two members of the group and we consistently observe an increase in the pre-resection group across the entire profile. Statistically Significant Changes in N-glycan Relative Intensities Pre- and Post Resection MALDI-TOF Mass Spectra of N-glycans Released from Blood Serum of Patient 549 Post-Resection MALDI-TOF mass spectra were acquired for each sample using a Applied Biosystems 4800 MALDI TOF/TOF Analyzer. Below is a representative spectrum demonstrating typical post-resection results from patient 549 Acknowledgements 2X This work was supported by grant No. GM24349 from the National Institute of General Medical Sciences, U.S. Department of Health and Human Services, and grant No. RR018942 from the National Center for Research Resources, a component of the National Institute of Health (NIH-NCRR) for the National Center for Glycomics and Glycoproteomics (NCGG) at Indiana University. 2X 2X 2X