Carbonyl-Reactive Tandem Mass Tag (TMT) Reagents for Mass Spectrometry-Based Quantitative Glycomics

Carbonyl-Reactive Tandem Mass Tag (TMT) Reagents for Mass Spectrometry-Based Quantitative Glycomics Sergei I. Snovida, 1 Rosa Viner, 2 John C. Rogers 1 1 Thermo Fisher Scientific, Rockford, IL; 2 Thermo Fisher Scientific, San Jose, CA

Overview Purpose: To demonstrate applications of the new carbonyl-reactive aminoxytmt reagent in quantitative analysis of carbohydrates by direct infusion and liquid chromatography-coupled mass spectrometry. Methods: released from standard glycoproteins, including several monoclonal antibodies, were individually labeled with the isobaric aminoxytmt reagents, and combined together for quantitative analysis by mass spectrometry. Results: Relative quantitation of the labeled glycans from different samples was done by measuring relative peak intensities of the TMT reporter ions at 2 or 3 levels. Introduction Aberrant glycosylation profiles may reflect abnormal physiological state of a cell, an organ, or an organism as a whole, and may be indicative of a disease state or cancer. Thus, glycans may be used as biomarkers. Additionally, many biotherapeutic drugs are glycoproteins and their activity and efficacy greatly depends on the type and extent of glycosylation. These compounds must be characterized in terms of their glycosylation profiles in both the development and quality control phases of drug development and manufacture. Accurate quantitation of glycans remains elusive due to the lack of a comprehensive selection of available standards, poor ionization efficiency of carbohydrates relative to other classes of biomolecules, and broad structural heterogeneity of glycomic samples. Recently, we introduced a set of isobaric carbonyl-reactive TMT reagents, Thermo Scientific aminoxytmt (Tandem Mass Tag ) Label Reagents (Figure 1), which can react with the reducing end of glycans to form a stable oxime product. The six compounds of the Thermo Scientific aminoxytmtsixplex Reagent Set have the same mass (i.e., isobaric) and chemical structure (carbonyl-reactive aminoxy group, spacer arm, and mass reporter). However, the specific distribution of 13 C and N isotopes on either side of the / fragmentation site in each reagent results in a unique reporter mass (126 131 Da) in the low region of 2 spectra. This set of reporter ions is used to measure the relative abundance of labeled molecules in a combined (multiplexed) sample representing six different treatment conditions, time points, or replicates. For glycobiology applications, the reagents enable quantitative profiling of glycoforms and discovery of glycan biomarkers; they provide improved ionization of glycans for increased sensitivity and increased analytical throughput by sample multiplexing. In this work, we showcase the use of these reagents for quantitative glycomics by combining our multiplexed TMT-based approach with HILIC LC- technique to enable more-sensitive analysis with improved glycome coverage. Methods Sample Preparation Bovine thyroglobulin (Sigma-Aldrich) and several monoclonal antibodies (Thermo Fisher Scientific) were reduced, alkylated, and digested with -grade trypsin (Thermo Fisher Scientific). Digest mixture was then treated with PNGase F (New England Biolabs) to release N-linked glycans. Following deglycosylation, released glycans were separated from peptides using Oasis HLB (Waters) solid phase extraction columns. After drying, glycan samples were labeled with aminoxytmt reagents (Thermo Fisher Scientific) according to manufacturer s protocol. Liquid Chromatography LC- experiments were performed in HILIC mode on a Thermo Scientific Accucore Amide column (2.6µm, Å; µm mm). Mobile phases were.1% formic acid in water (A) and.1% formic acid in acetonitrile (B). Separation was carried out with a 4 nl/min flowrate using different, sample-dependent gradients. Mass Spectrometry For direct infusion experiments, labeled glycan samples were dissolved in % acetonitrile solution containing µm NaOH. Samples were analyzed on Thermo Scientific Orbitrap Velos Pro ion trap and Thermo Scientific Orbitrap Fusion Tribrid mass spectrometers in the positive ion mode. Data Analysis All data was processed manually. FIGURE 2. Positive ion mode aminoxytmt-labeled high-m 1 9 8 7 6 4 3 2 1 1 9 8 7 6 4 3 2 1 unlabeled 138.17 347.26 226. 329.26 447.34 1 2 3 4 B (Na.34 aminoxytmt Reagent-labeled * 27.2 347.12 41.2.2 33.24 23.2 463.2 2 Carbonyl-Reactive Tandem Mass Tag (TMT) Reagents for Mass Spectrometry-Based Quantitative Glycomics

blf_ng_tmt6_2_1_infusion_ot 193_6res #1-36 RT:.-.13 AV: 36 NL:.8E T: FT + p ESI Full ms2 193.@hcd. [.-2.] 1 9 8 7 6 4 3 2 1 1. 126. 126. 127. 127. 128. 128. 129. 129. 13. 13. 131. 131. Results General Features of aminoxytmt-labeled Glycans Two types of the reagent are available, Thermo Scientific aminoxytmtzero Label Reagents, the light version of the reagent recommended for method development work, and the heavy, isobaric aminoxytmt sixplex set (Figure 1). When a labeled glycan is subjected to / fragmentation by, mass reporter region of the label molecule is cleaved, leading to an intense peak in the low region of / spectrum. For the aminoxytmtsixplex set, each member of the set produces a mass reporter ion with a unique, thus allowing for relative quantitation of glycoforms across up to six different samples (Figure 3). The carbonyl-reactive alkoxyamine group reacts rapidly with aldehydes and ketones to yield a stable oxime product, which is resistant to hydrolysis over a broad range of experimental conditions. Comparison of the fragmentation spectra of the sodiated precursors of unlabeled and aminoxytmt-labeled glycans (Figure 2) shows that the same structurally relevant product ions are produced for both the labeled and the unlabeled glycans, suggesting that structural information is not lost or diminished after labeling with the aminoxytmt reagent. Therefore, in addition to the mass reporter ions relevant for quantitation, information which can be derived from the / spectra of the unlabeled glycans is still present here. FIGURE 1. Chemical structure of aminoxytmt reagents and labeling reaction scheme. AminoxyTMT Reagent Structure AminoxyTMTsixplex Isobaric Reagents FIGURE 3. Workflow for quantitativ Std Lc-fluorescence Data #1 #2 Standard Reference Sample #3 #4 3_mAbs_12 T: IT + p 1 9 8 7 6 #4 3 2 1 126 127 128 129 13 131 TMT Reporter Ion Channels + Reaction: + glycan aminoxytmt reagent Stable oxime product FIGURE 2. Positive ion mode fragmentation of native unlabeled and aminoxytmt-labeled high-mannose glycans. 1 9 8 7 6 4 3 2 1 1 9 8 7 6 4 3 2 1 136.61 NL: 1.16E4 RNAseB_NG_2ug_prot_ unlabeled eq_ml_641#9 RT:.1 [M+2Na] 2+ AV: 1 T: IT + p ESI Full [M+Na] + ms2 64.@hcd. [.-2.] 17.69 874.2 671.43 833.2 1.61 447.34 Y (Na +,2 ) A 97.2.34 771.2 16.61 64.68 347.26 994.61 226. 99. 138.17 329.26 13.7 14.7 136.44 NL:.17E3 blf_rnaseb_btg_tmt_pos_78 # RT:.9 AV: 1 T: 78.@hcd. 671.28 4.44 [.-2.] 1.36 C (Na + ) aminoxytmt Reagent-labeled * 3.28 833.32 97.36 [M+ Na-reporter-CO] + 979.4,2 6.28 A 27.2 689.28 16.48 347.12.24 817.36 33.24 41.2 1211.2 23.2 463.2 1272.2 14.68 1 2 3 4 6 7 8 9 1 11 12 13 14 Trap- 64. Trap- 779.9 Thermo Scientific Poster Note PN-64133-AS-EN-614S 3

blf_ng_tmt6_2_1_infusion_ot 193_6res #1-36 RT:.-.13 AV: 36 NL:.8E T: FT + p ESI Full ms2 193.@hcd. [.-2.] 1 9 8 7 6 4 3 2 1 1. 126. 126. 127. 127. 128. 128. 129. 129. 13. 13. 131. 131. FIGURE 3. Workflow for quantitative glycomics multiplexed experiment. Lc-fluorescence Data Unknown Samples Standard Reference Sample Std #1 #2 #3 #4 # + + + + + TMT 126 TMT 127 TMT 128 TMT 129 TMT 13 TMT 131 RT:.39-99.74 1 9 8 7 6 4 47. 47.92 76.94 76.49 77.9 69.73 69.39 63.62 77.28 63.49 69.87 69.27 Std #1 #2 #3 126 127 128 129 13 131 #4 TMT Reporter Ion Channels + 3 77.67 66.18 61. 77.92 2 76.2 78. 7.3 6.4 3_mAbs_123123 #4 RT:.11 AV: 1 NL: 2.41E6 48.73 6. 78.7 T: IT + p ESI Full ms [7.-.] 1 48.89 78.97 893.84 1. 79. 74.3 83.66 27.87 34.1 38. 43.87 46.49 Sixplex Sample 3 4 6 7 8 9 Time (min) 8 7 / 6 #4 3 2 882.84 91.84.93 Actual ratios 1 94.84 963.92 982.84 82.84 873.34 792.34 829. 14.18 1:2:::2:1 8.67 91.76 4.34 991.84 163.84 198.9 114.1 1168.18 1198.1 1226.1 8 9 1 11 1 12 1.4E 3.9E of native unlabeled and 36.61 NL: 1.16E4 RNAseB_NG_2ug_prot_ eq_ml_641#9 RT:.1 [M+2Na] 2+ AV: 1 T: IT + p ESI Full [M+Na] + ms2 64.@hcd. [.-2.] 17.69 1.61,2 A 16.61 1 13.7 14.7 36.44 NL:.17E3 blf_rnaseb_btg_tmt_pos_78 # RT:.9 AV: 1 T: 78.@hcd. 4.44 [.-2.] C (Na + ) [M+ Na-reporter-CO] +,2 A 16.48 1211.2 14.68 1272.2 11 12 13 14 Trap- 64. Trap- 779.9 Multiplexing Glycomic Samples - Overview In a typical experiment, up to six separate samples may be analyzed together, which allows lower overall analysis time and minimizes the variations in quantitative reproducibility, as the number of independent sample handling steps is reduced in this workflow (Figure 3). After labeling with aminoxytmt reagents, unreacted reagents are quenched with acetone and samples are combined. An additional clean-up step using HILIC solid phase extraction material is used to separate the quenched reagent from the labeled glycans. During analysis, precursor ions corresponding to the glycoforms of interest are identified and are subjected to / analysis by fragmentation. Relative peak intensities of the reporter ions observed in the / spectra are proportional to the relative abundance levels of the selected glycoform in different samples in the set. Several peaks observed in the low region of / spectra of the labeled glycans may interfere with accurate quantitative analysis if a low resolution mass spectrometer is used (Figure 3). These peaks are some of the product ions of HexNAc fragmentation, and are thus present in / spectra of most glycoforms. Although this may be an issue for low resolution instruments, these interferences are easily resolved and do not pose a problem for accurate quantitation at the lowest resolution settings of a Thermo Scientific Orbitrap mass spectrometer. If a Velos Pro ion trap mass spectrometer is used for the analysis, application of a targeted 3 method (Figure 4) solves this interference problem. First, precursor of interest is isolated and fragmented using CID or fragmentation mode. In the second step, abundant Y 1 ions, which still contain intact aminoxytmt label, are isolated for 3. 3 spectra contain clean mass reporter ions and their ratios are now as expected for the sample set. Multiplexing Glycomic Samples - Examples Several experiments showcasing the use of these reagents for quantitative analysis of glycans were performed using antibodies as glycan sources. In the first experiment (Figure ), 1 µg of two anti c-myc monoclonal antibodies, each coming from a different source, were separately deglycosylated using PNGase F glycosidase, glycans were isolated and each sample was labeled with a different heavy version of the isotopic aminoxytmt reagent. After quenching and clean-up, the samples were combined and analyzed by direct infusion using a Velos Pro mass spectrometer. Anti-c-Myc hybridoma 1 8 6 4 2 Extracted glycan profiles 1 2 3 IgGs 1 8 6 4 2 Re Mouse Culture Relative quantitation of the three by comparing relative peak inten spectra for each glycoform. After produced in the cell culture cont than the antibodies obtained from 4 Carbonyl-Reactive Tandem Mass Tag (TMT) Reagents for Mass Spectrometry-Based Quantitative Glycomics

1 9 8 7 6 4 3 2 1 1 9 8 7 6 4 3 2 1 122. 122.92 126. 126.8 127. 127. 128. 129. 13. 13. 131. 131. 132.92 122 124 126 128 13 132 134 136 NL: 1.3E4 btg_ng_native_oxytmt_1_2 hilic_acnsamp le_1_1ul#6214-689 RT:.2-8.62 AV: 42 F: IT + p NSI d Full ms2 891.63@hcd8. [1.-2.] 1to1_mix_native_TMT_pos #18 RT:.11 AV: 1 NL: 1.82E T: IT + p ESI E Full ms [.-2.] 893.92 1 9 8 7 6 4 3 2 1 91.88 94.92 963.96 831.6 86.92 933.88 946.92 114.92 128.4 176.2 113.84 84 86 88 9 92 94 96 98 1 12 14 16 18 11 112 ltiplexed experiment. 893.84 Unknown Samples 4 91.84 #2 #3 #4 # 94.84 + + + TMT 128 TMT 129 TMT 13 TMT 131 Sixplex Sample 963.92 982.84.93 FIGURE 4. Targeted 3 acquisition approach using a Velos Pro ion trap instrument. RT:.39-99.74 14.18 91.76 4.34 163.84 114.1 991.84 1198.1 198.9 1226.1 1168.18 1 9 8 7 6 4 3 2 1 47. 47.92 3 4 6 7 8 9 Time (min) 121.92 124. 124.92 131.92 134. 1.92 Actual ratios 1:2:::2:1 7.3 6.4 48.73 6. 128. 129. 63.49 61. 63.62 69.39 69.27 69.73 69.87 76.49 76.94 77.9 77.28 77.67 66.18 77.92 48.89 78.97. 79. 74.3 83.68 88.41.66 27.87 34.1 46.49 84.94 38. 43.87 89.22 76.2 78. 78.7 1.4E4 3.9E2 97. DDA/targeted 98. NL: 9.19E6 Base Peak btg_ng_native_oxy TMT_1_2 HILIC_ ACNsample_1_1 ul_14219224437 1 8 6 4 2 1 8 6 4 2 1 8 6 4 2 137.92 24. 128. 274.9 891.44 99.8 682.36 7.8 124.16 1336.68 4.2 23.4 11.88.72 1723.92 1916.8 23.34 Y 1 -ion 726.2 2 ( or CID) 366.17 669.43 888.2.61.34 66.43 872.2 1212.7 18.78 77.87 1723.88 93.2 12.96 clean reporter ions 3 () [M+3H] 3+ 33.17 367.9 668. 891.44 1246.3.26 19.61 1422.4 1669.88 1922.8 2 4 6 8 1 12 14 16 18 2 In another example (Figure 6), 1 deglycosylated using PNGase F g pool was divided into two equal pa the heavy isobaric aminoxytmtsix the individual samples were comb three separate samples in a duplic infusion to reveal that two out with respect to glycosylation profile antibody having substantially highe FIGURE 6. Quantitative compari antibodies with replicates. NL: 7.4E4 btg_ng_native_oxytmt_1_2 hilic_acnsamp le_1_1ul#6214-689 RT:.2-8.62 AV: 42 F: IT + p NSI d Full ms2 891.63@hcd8. [1.-2.] NL: 3.88E2 btg_ng_native_oxytmt_1_2 HILIC_AC Nsample_1_1uL_14219224437#1196-12461 RT:.2-8.96 AV: 147 F: IT + p NSI d Full ms3 891.@hcd8. 23.3@hcd1. [1.-2.] NL: 1.93E6 btg_ng_native_oxytmt_1_2 HILIC_AC Nsample_1_1uL_14219224437#1196-12461 RT:.-81. AV: 148 T: IT + p NSI E Full ms [4.-2.] NL: 3.88E2 btg_ng_native_oxytmt_1_2 HILIC_AC Nsample_1_1uL_14219224437#1196-12461 RT:.2-8.96 AV: 147 F: IT + p NSI d Full ms3 891.@hcd8. 23.3@hcd1. [1.-2.] TMT 126 TMT 129 TMT 127 TMT 13 TMT 128 TMT 9 1 11 1 12 123.8 1. 132. 133.8 134. 1.17 136.8 3_mAbs_1231 T: IT + p E 1 9 FIGURE. Comparing glycosylation profiles of two monoclonal antibodies from different sources. Anti-c-Myc hybridoma IgGs 1/1 Deglycosylate, label, mix, clean 1/1 GF G1F 882.92 982.92.96 873.44 913.4 993.92 144.92 163.92 1122. 1137.44 G2F Combined Sample 8 7 6 4 3 2 1 Extracted glycan profiles Reporter ion relative intensities 1 1 1 8 128.12 6.24 9.36 1118.44 931.36 1136.44 NL: 7.76E2 native_tmt128_131_pos_8 94# RT:.1 AV: 1 T: 894.@hcd. [.-2.] 8 6 4 2 8 6 4 2 4 2 1 8 6 4 2 128.12 32.2 989.4 139.2 1238.48 1384.6 33.24 388.16 66.24 786.32 1482.64 163.72 1743.2 19.16 128.2 NL: 2.E2 native_tmt128_131_pos_97 9.36 # RT:.11 AV: 1 T: 177.44 9.@hcd. [.-2.].24 168.4 33.24 11.44 388.16 728.28 931.36 1471.6 32.24 786.32 68.24 14.6 46.6 1644.68 46.16 1792.8 1927.48 1 2 3 1 2 3 1 8 6.24 874.32 9.36 177.44 1239.48 1442.6 NL: 1.79E1 native_tmt128_131_pos_1 6#4 RT:.9 AV: 1 T: 6.@hcd. [.-2.] Mouse Culture 4 2 388.16 1313.2.76 933.36 728.28 1633.64 1181.48 68.24 168.8 327.24 62.64 186.76 32.2 12.88 2 4 6 8 1 12 14 16 18 2 Relative quantitation of the three main glycoforms, GF, G1F, and G2F, was performed by comparing relative peak intensities of the mass reporter peaks in the / spectra for each glycoform. After signal normalization, we found that the antibody produced in the cell culture contained higher amounts of the G1F and G2F glycoforms than the antibodies obtained from the mouse. Thermo Scientific Poster Note PN-64133-AS-EN-614S

1to1_mix_native_TMT_pos #18 RT:.11 AV: 1 NL: 1.82E T: IT + p ESI E Full ms [.-2.] 893.92 1 9 8 7 6 4 3 2 91.88 1 94.92 963.96 831.6 86.92 933.88 946.92 114.92 128.4 176.2 113.84 84 86 88 9 92 94 96 98 1 12 14 16 18 11 112 3_mAbs_123123_894 #1 RT:.1 AV: 1 NL:.69E3 T: IT + p ESI Full ms2 893.8@hcd. [.-.] 126. 1 128.34 131.34 13.34 9 127.34 129.34 8 7 6 4 3 2 124. 1 1. 3_mabs_123123_9 #21132. RT:.24 AV: 1 NL:.48E3 122. 123. T: IT + p ESI Full ms2 9.2@hcd. 133. [.-.] 134. 1.17 126. 122 123 124 1 126 127 128 129 13 1 131 132 133 134 1 129.34 9 8 7 6 4 127.34 128.34 13.34 131.34 3 2 1 124.17 1. 132. 122. 123. 133. 134. 1.17 122 123 124 1 126 127 128 129 13 131 3_mabs_123123_6 132 133 #8 RT: 134.1 AV: 11 NL: 1.22E3136 T: IT + p ESI Full ms2 6.2@hcd. [.-.] 126. 1 9 129.34 8 7 6 4 3 2 127. 128.34 13.34 131.34 1 124.17 1. 122. 123. 132. 133. 134.17 1.17 136. 122 123 124 1 126 127 128 129 13 131 132 133 134 1 136 d 891.44 [M+3H] 3+ In another example (Figure 6), 1 µg of three different monoclonal antibodies were deglycosylated using PNGase F glycosidase, glycans were isolated, and each sample pool was divided into two equal parts. Each part was labeled with a different version of the heavy isobaric aminoxytmtsixplex reagent, and after a quenching and clean-up, the individual samples were combined to yield a single sixplex sample consisting of three separate samples in a duplicate. The sample was then analyzed by direct infusion to reveal that two out of three different antibodies analyzed were identical with respect to glycosylation profiles of the three main glycoforms, with the third antibody having substantially higher abundance of the G1F and G2F glycoforms. NL: 1.93E6 btg_ng_native_oxytmt_1_2 HILIC_AC Nsample_1_1uL_14219224437#1196-12461 RT:.-81. AV: 148 T: IT + p NSI E Full ms [4.-2.] 99.8 682.36 7.8 124.16 1336.68 23.4 11.88.72 1723.92 1916.8 23.34 Y 1 -ion 2 ( or CID) FIGURE 6. Quantitative comparison of glycosylation of several monoclonal antibodies with replicates. NL: 7.4E4 btg_ng_native_oxytmt_1_2 hilic_acnsamp le_1_1ul#6214-689 RT:.2-8.62 AV: 42 F: IT + p NSI d Full ms2 891.63@hcd8. [1.-2.] 726.2 669.43 888.2.61.34 66.43 872.2 1212.7 18.78 77.87 1723.88 93.2 12.96 er 3 () NL: 3.88E2 btg_ng_native_oxytmt_1_2 HILIC_AC Nsample_1_1uL_14219224437#1196-12461 RT:.2-8.96 AV: 147 F: IT + p NSI d Full ms3 891.@hcd8. 23.3@hcd1. [1.-2.] 668. 891.44 1246.3.26 19.61 1422.4 1669.88 1922.8 6 8 1 12 14 16 18 2 GF G1F TMT 126 TMT 129 TMT 127 TMT 13 TMT 128 TMT 131 Combined Sample GF 3_mAbs_123123 #4 RT:.11 AV: 1 NL: 2.41E6 T: IT + p ESI Full ms [7.-.] 893.84 1 9 8 7 G1F 6 4 3 2 G2F 882.84 91.84.93 1 94.84 963.92 982.84 82.84 873.34 792.34 829. 14.18 8.67 91.76 4.34 991.84 163.84 198.9 114.1 1168.18 1198.1 1226.1 8 9 1 11 1 12 es 1 8 6 4 2 1 8 6 4 2 1 8 6 4 2 882.92 982.92.96 873.44 913.4 993.92 144.92 163.92 1122. 1137.44 G2F 9.36 1118.44 NL: 7.76E2 native_tmt128_131_pos_8 94# RT:.1 AV: 1 T: 128.12.24 894.@hcd. 931.36 [.-2.] 1136.44 989.4 139.2 32.2 1238.48 1384.6 33.24 388.16 66.24 786.32 1482.64 163.72 1743.2 19.16 128.2 NL: 2.E2 native_tmt128_131_pos_97 9.36 # RT:.11 AV: 1 T: 177.44 9.@hcd. 128.12 [.-2.].24 11.44 388.16 728.28 931.36 1471.6 32.24 786.32 68.24 14.6 46.6 168.4 33.24 1644.68 46.16 1792.8 1927.48 NL: 1.79E1 native_tmt128_131_pos_1 177.44 6#4 RT:.9 AV: 1 T: 1442.6 6.@hcd. [.-2.] 9.36.24 874.32 1239.48 388.16.76 1313.2 933.36 728.28 68.24 1181.48 1633.64 168.8 327.24 62.64 186.76 32.2 12.88 2 4 6 8 1 12 14 16 18 2 F, G1F, and G2F, was performed porter peaks in the / n, we found that the antibody ts of the G1F and G2F glycoforms Conclusion Increase sample analysis throughput with aminoxytmt reagents Overall improvement in sensitivity with aminoxytmt label approach Structural elements are preserved in labeled glycans LC-UV/fluorescence and TMT-based quantitation can be complementary Ideal for analysis of biotherapeutics and biomarker discovery Acknowledgements We would like to thank the following colleagues and collaborators for critical advice and helpful discussions: Dr. Ryan Bomgarden, Dr. Chris Etienne, Dr. Kay Opperman, Dr. Venky Shivalingappa, and Dr. Julian Saba (Thermo Fisher Scientific); Dr. Kay-Hooi Khoo, Dr. Ming-Yi Ho, Dr. Shui-Hua Wang, and Dr. Chia-Wei Lin (Academia Sinica, Taipei, Taiwan); Dr. Ian Pike and Dr. Karsten Kuhn (Proteome Sciences); Dr. Bernhard Küster, Dr. Hannes Hahne, and Dr. Patrick Neubert (Technische Universität München, Munich, Germany); Dr. Lingjun Li, Dr. Xuefei Zhong, and Dr. Yan Liu (University of Wisconsin, Madison, WI, USA); Dr. Yehia Mechref, Shiyue Zhou, and Yunli Hu (Texas Tech University, Lubbock, TX, USA); Dr. Shonali Paul and Dr. Sanjib Meitei (Premier Biosoft). PO64133-EN 614S 6 Carbonyl-Reactive Tandem Mass Tag (TMT) Reagents for Mass Spectrometry-Based Quantitative Glycomics

www.thermoscientific.com 214 Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization. Tandem Mass Tag and TMT are registered trademarks of Proteome Sciences plc. Sigma-Aldrich is a registered trademark of Sigma-Aldrich Co. LLC. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Africa +43 1 333 34 Australia +61 3 97 43 Austria +43 81 282 26 Belgium +32 3 73 42 41 Canada +1 8 3 8447 China 8 81 118 (free call domestic) 4 118 Denmark + 7 23 62 6 Europe-Other +43 1 333 34 Finland +8 9 3291 2 France +33 1 6 92 48 Germany +49 613 48 114 India +91 22 6742 9494 Italy +39 2 91 Japan +81 3 91 Latin America +1 61 688 87 Middle East +43 1 333 34 Netherlands +31 76 79 New Zealand +64 9 98 67 Norway +46 8 6 468 Russia/CIS +43 1 333 34 Thermo Fisher Scientific, San Jose, CA USA is ISO 91:28 Certified. Singapore + 6289 119 Spain +34 914 8 9 Sweden +46 8 6 468 Switzerland +41 61 716 77 UK +44 1442 23 USA +1 8 32 42 PN-64133-EN-614S