Solid-phase extraction of N-linked glycans and glycosite-containing peptides (NGAG) for comprehensive characterization of glycoproteins
A novel chemoenzymatic method termed solid phase extraction of N-linked Glycans And Glycosite-containing peptides (NGAG) for the simultaneous analysis of N-glycans, glycosite-containing peptides, and intact N-glycopeptides with site-specific glycosylation information.
Sample Processing Protocol
The NGAG method includes seven steps. 1) Protease digestion and lysine modification: protein samples were digested into peptides, followed by guanidination to block the ε-amino groups on the side chain of lysine residues. 2) Conjugation to solid support: peptides were covalently conjugated to the aldehyde-functionalized solid support (in the presence of sodium cyanoborohydride) via their N-termini (α-amino groups) through reductive amination. 3) Carboxyl group modification: aspartic acid (D), glutamic acid (E), and peptide C-termini were modified by aniline through their carboxyl group modification; meanwhile, sialic acids on the sialylated glycopeptides were also modified by aniline to facilitate mass spectrometric detection of sialylated glycans. 4) N-Glycan release: N-linked glycans were released from the solid support via PNGase F digestion, in parallel with conversion of asparagine residues (N) at N-linked glycosites to aspartic acid (D) residues via deamination. 5) Glycosite-containing peptide release: Glycosite-containing peptides with aspartic residues at N-glycosites were specifically released from solid phase via Asp-N digestion. Asp-N specifically cleaves peptide bonds N-terminal to aspartic acid residues. However, because all original aspartic acid residues of the peptides were modified with esterification, only newly generated aspartic acid residues at the N-glycosites after N-glycan release are cleaved by Asp-N. 6) Identification: the released N-glycans and glycosite-containing peptides were identified by mass spectrometry. 7) The intact N-glycopeptides were then determined from the oxonium ion-containing MS/MS spectra from enriched intact glycopeptides or from global proteomic data based on the selection of glycopeptide precursor ions, peptide+HexNAc (or peptide) fragment ions, as well as the b- and y-ions using the glycopeptide candidate database (comprised of identified glycans and glycosite-containing peptides).
Data Processing Protocol
All LC-MS/MS data from human and bovine resources were searched against RefSeq human protein databases51 (downloaded from NCBI website July 29th, 2013) and bovine fetuin sequence by MaxQuant52 (v18.104.22.168), respectively. For global proteome data (tryptic peptide), the search parameters were set as follows: up to two missed cleavage were allowed for trypsin digestion, 20ppm and 6ppm precursor mass tolerance for first and main search, respectively; Carbamidomethylation (C) was set as a static modification and oxidation (M) was set as a dynamic modification; two modifications with “Arg 10” and “Lys6” were selected as heavy labels for mixed peptides from OVCAR-3 cells; five modifications per peptide and a minimum of six amino-acid length were considered for peptide identification. All other settings were set as default values and the results were filtered with a 1% FDR. For SPEG glycosite-containing peptides data, deamination (N) was added as one additional dynamic modification. In order to search LC-MS/MS data of glycosite-containing peptides extracted by NGAG, both human and bovine fetuin databases were first modified by replacing all potential N-glycosylation sites (N#-X-S/T motif, X is any amino acid except proline) with “U” (not existing in normal protein sequences) to facilitate the search parameter setting and N-linked glycosite-containing peptide identification. Then an enzyme “trypsin + U” was created and added to the enzyme list which was assigned to specifically cleave peptide bonds at the C-terminal side of lysine/arginine (no cleavage at KP and RP) and at the N-terminal side of “U” (168.9642Da). The MaxQuant search parameters were set as follows: up to two missed cleavages were allowed for “trypsin+U” digestion, 20ppm and 6ppm precursor mass tolerance for first and main search, respectively; Carbamidomethylation (C, +57.0215Da) was set as a static modification; U->D (U, -53.9373Da), U->N (U that is not at the N-termini, -54.9213Da), guanidination (K, +42.0218Da), aniline label (D and E that are not at the N-termini, protein C-termini and K and R that are at any C-termini, +75.0473Da), two aniline labels (D and E that are at the protein C-termini, +150.0946Da), guanidination + aniline label (K at any C-termini, +117.0691Da) and oxidation (M, +15.9949Da) were set as dynamic modifications; six modifications per peptide and a minimum of six amino-acid length were considered for peptide identification. All other settings were set as default values and the results were filtered with a 1% FDR. For glycosite-containing peptide data from the OVCAR-3 cell line which included SILAC peptides, K6+guanidination label (K, +48.0419Da), K6+guanidination+aniline label (K at any C-termini, +123.0892Da) and R10+aniline label (R at any C-termini, +85.0555Da) were added as additional dynamic modifications based on the parameters used for the global proteome data described above. Two modifications with “Arg 10” and “Lys6” were selected as heavy labels.
Sun S, Shah P, Eshghi ST, Yang W, Trikannad N, Yang S, Chen L, Aiyetan P, Höti N, Zhang Z, Chan DW, Zhang H. Comprehensive analysis of protein glycosylation by solid-phase extraction of N-linked glycans and glycosite-containing peptides. Nat Biotechnol. 2015 Nov 16 PubMed: 26571101
Shu J, Dang L, Zhang D, Shah P, Chen L, Zhang H, Sun S. Dynamic analysis of proteomic alterations in response to N-linked glycosylation inhibition in a drug-resistant ovarian carcinoma cell line. FEBS J. 2019 PubMed: 30884134