Supporting information

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1 Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 2014 Supporting information Glycan Reductive Isotope-coded Amino Acid Labeling (GRIAL) for Mass Spectrometry-based Quantitative Glycomics Yan Cai a,b, Jing Jiao a,b, Zhichao Bin b, Ying Zhang a,b *, Pengyuan Yang b and Haojie Lu a,b * a Shanghai Cancer Center and Key Laboratory of Glycoconjuates Research Ministry of Public Health, Fudan University, Shanghai , P. R. China b Institutes of Biomedical Sciences and Department of Chemistry, Fudan University, Shanghai, , P. R. China *Corresponding author, Tel.: ; fax: luhaojie@fudan.edu.cn; ying@fudan.edu.cn.

2 Experimental section Materials and reagents Maltoheptaose (DP7, 95%) was purchased from Hayashibara Biochemical Laboratories (Okayama, Japan). The biantennary complex glycan NA2 (>90%) was purchased from Ludger Ltd (Oxford, UK). Dextran (analytical standard, for GPC, 1000), 2,5-Dihydroxybenzoic acid (DHB), L-Arginine ( 12 C 6 -Arg, 98%), trifluoroacetic acid (TFA), ammonium bicarbonate (ABC), sodium cyanoborohydride (NaBH 3 CN), asialofetuin from fetal calf serum (ASF), albumin from chicken egg white (OVA) and ribonuclease B from bovine pancreas (RNase B) were obtained from Sigma- Aldrich (St. Louis, MO, USA). L-Arginine ( 13 C 6 -Arg, 99%) was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). Peptide N-glycosidase (PNGase F, 500 U/μL) was obtained from New England Biolabs (Ipswich, MA, USA). Centrifugal filters with MWCO of 3 kda and 10 kda were obtained from Millipore (Bedford, MA, USA). HPLC-grade methanol and acetonitrile (ACN) were purchased from Merck (Darmstadt, Germany). Analytical-grade acetic acid was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Distilled water was purified by a Milli-Q system (Milford, MA, USA). Preparation of N-glycans from model glycoproteins and human serum For preparation of N-glycans from model glycoproteins, 0.5 mg of RNase B, OVA and ASF were dissolved in 25 mm ABC buffer (ph 8.0), respectively. After denaturation by heating at 100 C for 10 min, PNGase F solution (0.5 μl) was added to the glycoprotein solution. The deglycan reaction was carried out at 37 C for 16 h and the released glycans were recovered by ultrafiltration through a membrane with MWCO of 3 kda. The glycans were further purified over a Carbograph column (Extract-Clean SPE Carbo 150 mg; Grace Division Discovery Science; Deerfield, IL). The collected glycans were then lyophilized and resuspended in ultrapure water for further use. Human serum samples were collected from three patients diagnosed with colorectal cancer (CRC) and three healthy control individuals at Fudan University Shanghai Cancer Center. For preparation of N-glycans from human serum, human serum was treated according to the protocol reported previously with some modifications. Briefly, 10 μl of serum from pooled samples was diluted with 190 μl of 25 mm ABC buffer (ph 8.0) containing 8 M urea and 10 mm dithiothreitol. After incubation at 37 C for 60 min, iodoacetamide (20 mm final concentration) was added. The sample was incubated at room temperature for 60 min in the dark and then the low-molecularweight molecules (reagents, salts) were removed by using a centrifugal filter (MWCO, 3 KDa). The collected proteins were washed with 200 μl of 25 mm ABC buffer for three times and dissolved in 190 μl of 25 mm ABC buffer. PNGase F (1 μl) was added and the sample was incubated at 37 C overnight. The released N-glycans were recovered by ultrafiltration (MWCO, 10 KDa) and further purified over a Carbograph column. The collected glycans were then lyophilized and resuspended in ultrapure water. One-tenth of the glycans were used for further analysis.

3 Labeling the reducing ends of glycans with amino acids DP7 and NA2 were individually dissolved in distilled water at a concentration of 1 mg/ml as a storage solution and diluted to the proper concentration as needed. Arg( 12 C 6 ) and Arg( 13 C 6 ) were indivedually dissolved in distilled water at a concentration of 100 mg/ml. Prior to the labeling, all of the samples were lyophilized and redissolved in 10 μl of methanol-acetic acid (98:2, v/v) containing 0.1 mg/ml NaBH 3 CN. Then 1 μl of Arg( 12 C 6 ) or Arg( 13 C 6 ) was added and then the sample solution was incubated at 65 C for at least 3.5 h. The labeled glycans were purified with Carbon Micropipette Tips (Sigma-Aldrich, St. Louis, MO) and subjected to MALDI-TOF MS analysis. MALDI mass spectrometric analysis DHB matrix solution was prepared at a concentration of 10 mg/ml in ACN/water (50:50, v/v) containing 0.1% TFA. The sample solution (0.5 μl) was deposited on the target with DHB matrix (0.5 μl) by the dried-droplet method for MALDI-TOF MS analysis. All MALDI-TOF MS and MS/MS experiments were performed in positive ion reflection mode on a 5800 Proteomics Analyzer (Applied Biosystems, Framingham, MA, USA) equipped with a Nd:YAG laser (355 nm), a repetition rate of 400 Hz, and an acceleration voltage of 20 kv. The laser intensity was set to 4500±200 a.u. to ensure good signal intensity without generation of extensive noise shots were accumulated for each spectrum. MS/MS spectra were interpreted manually with the assistance of the GlycoWorkbench software. Figure S1. MALDI-TOF mass spectrum of (A) DP7 labeled with Arg( 13 C 6 ) and the mixture of native DP7 and Arg( 12 C 6 ) labeled DP7 in molar ratio of (B) 1:5 and (C) 1:10. (A) (B) (C)

4 Figure S2. MALDI-TOF mass spectra of N-glycans from RNase B. (A) native, (B) labeled with Arg( 12 C 6 ), (C) labeled with Arg( 13 C 6 ). Figure S3. MALDI-TOF mass spectra of N-glycans from OVA. (A) native, (B) labeled with Arg( 12 C 6 ), (C) labeled with Arg( 13 C 6 ).

5 Figure S4. MALDI-TOF mass spectrum of an equimolar mixture of native Dextran and Dextran labeled with Arg( 12 C 6 ). Figure S5. HILIC-MS analysis of an equimolar mixture of Arg( 12 C 6 )-labeled and Arg( 13 C 6 )- labeled DP7. Conditions: column: TSKgel Amide-80 3 µm (4.6 mm ID 15 cm L); Eluents: Ammonium formate (5 mm, ph 4.5)/ACN=70/30; Flow rate: 0.4 ml/min; Injection volume: 10 µl; Temperature: 25. Figure S6. MALDI-TOF mass spectra of the mixture of Arg( 12 C 6 ) labeled DP7 and Arg( 13 C 6 ) labeled DP7 in a molar ratio of (A) 1:1, (B) 1:5, and (C) 1:10.

6 Figure S7. MALDI-TOF mass spectra of an equimolar mixture of Arg( 12 C 6 )-labeled and Arg( 13 C 6 )- labeled N-glycans from (A) RNase B and (B) OVA. Figure S8. Dynamic range and accuracy of quantitation of NA2 and N-glycans from RNase B (Man 5, Man 6 and Man 8). Glycans labeled with Arg( 12 C 6 ) and Arg( 13 C 6 ) were mixed in known molar ratios (1:10, 1:5, 1:1, 5:1, 10:1) and each experiment was repeated three times.

7 Figure S9. MALDI-TOF/TOF tandem mass spectra of (A) DP7 and (B) glycan isomers. (A) Native DP7 at m/z (top), DP7 labeled with Arg( 12 C6) at m/z (middle), DP7 labeled with Arg( 13 C 6 ) at m/z (bottom). (B) A pair of isomeric glycans labeled with Arg( 12 C 6 ) at m/z The top is from OVA with a hybrid structure, and the bottom is from ASF with a complex structure. Figure S10. MALDI-TOF mass spectrum of N-glycans from human serum. The N-glycans from normal samples were labeled with Arg( 12 C 6 ), and the N-glycans from CRC samples were labeled with Arg( 13 C 6 ). The samples were mixed in a molar ratio of 1:1 and subjected to MALDI-TOF MS analysis. Native DP7 was used as the spiked standard before labeling.

8 Figure S11. MALDI-TOF mass spectra of a sialylated glycan labeled with Arg( 12 C 6 ) in the (A) positive and (B) negative mode. (A) (B) Table S1. GRIAL for relative quantitation of N-glycans from normal and CRC human serum., N-acetyl glucosamine;, mannose;, galactose;, sialic acid;, fucose. No. Composition Light m/z Heavy m/z Ratio (CRC/normal) CV Change % No % No % No % No % Up % No % No % No

9 % Up % Up % No % No % No % Up % Up % Up % Up % Up % No % No % Up % Up % Up % No % Up

10 % No % No % No % No % Up % No