Nature Biotechnology: doi: /nbt Supplementary Figure 1

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1 Supplementary Figure 1 The timeline of the NGAG method for extraction of N-linked glycans and glycosite-containing peptides. The timeline can be changed based on the number of samples.

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3 Supplementary Figure 2 Identification of N-linked glycans, glycosite-containing peptides, and intact glycopeptides from bovine fetuin using the NGAG method. (a) MALDI-TOF-MS spectrum of fetuin glycans. The possible glycan structures of the top 13 peaks are shown in the spectrum. (b) MS/MS spectrum of a glycosite (Asn156)-containing fetuin peptide. (c) MS/MS spectrum of an intact glycopeptide at glycosite Asn156 with a HexNAc5Hex6 (N5H6) glycan attached. Oxonium ions (highlighted in green) were used to select the glycopeptide spectra from the global data, and the accurate masses of the precursor ion and peptide+hexnac/peptide (highlighted in blue) fragment ions as well as b/y fragment ions were used to identify the intact glycopeptide. (d) Site-specific heterogeneity of bovine fetuin. N: HexNAc; H: Hexose; F: Fucose; A: Sialic acid. The intact glycopeptides were identified from triplicate LC-MS/MS analysis.

4 Supplementary Figure 3 MS/MS spectra of the glycosite-containing peptides (deglycosylated peptides) identified from bovine fetuin. (a) The peptide N # CSVR with glycosite 99. (b) The peptide N # DSR with glycosite indicates the cleavage site by trypsin. The LC- MS/MS data was analyzed by Proteome Discoverer software (Thermo Fisher Scientific) allowing trypsin (semi) as the enzyme and at least 4 amino acids for peptide identification.

5 Supplementary Figure 4 MALDI-TOF-MS spectra of N-glycans isolated from OVCAR-3 cells using NGAG and the possible N-glycan structures with the composition matched to the glycan masses.

6 Supplementary Figure 5 The detection of glycans with 1Da mass difference by LC-MS using a Q-Exactive mass spectrometer. Two pairs of glycans with 1Da mass difference were used as the examples. These glycans with 1Da differences can be clearly distinguished based on their isotopic masses and retention time (which are mainly determined by the number of aniline-modified sialic acids) in LC-MS analysis.

7 Supplementary Figure 6 Quantification of glycosite-containing peptides between duplicate NGAG isolations.

8 Supplementary Figure 7 Reproducible analyses of N-glycans and glycosites using the NGAG method. (a) The number of identified glycans from three NGAG isolations. Each glycan sample was analyzed once by LC-MS/MS. (b) Label-free quantification of the identified glycans between two isolations from the same amount of fetuin. (c) Relative quantification of glycositecontaining peptides (itraq labeling method) isolated from different amounts of fetuin. (d) Label-free quantification of intact glycopeptides between replicate analyses of tryptic peptides from fetuin. (e) Comparison of identified OVCAR-3 cell glycans among three NGAG isolations. (f) Comparison of identified glycosite-containing peptides from OVCAR-3 cells among three NGAG isolations. (g) Comparison of identified glycosite-containing peptides among three LC-MS/MS injection of the same sample isolated from OVCAR- 3 cells. Each glycan or glycosite-containing peptide sample was analyzed once by LC-MS/MS.

9 Supplementary Figure 8 Liquid chromatography profiles of the glycans of OVCAR-3 cells from triplicate isolations using the NGAG method. The base peak profiles of the raw files of the triplicate isolations are displayed in Xcalibur.

10 Supplementary Figure 9 Liquid chromatography profiles of glycosite-containing peptides of OVCAR-3 cells from (a) 3 LC-MS/MS replicates and (b) 3 isolation replicates using the NGAG method. The base peak profiles of the raw files of triplicate isolations are displayed in Xcalibur.