IGP data were searched with our inhouse developed glycopeptide
analysis software, GPQuest 2.03 (link),26 (link) Any
peptide-spectrum match (PSM) without glycan annotation and a glycan
intensity ratio less than 20% were filtered out with consideration of a
false discovery rate (FDR) less than 1%.
Deglycosylated peptides and global peptides were searched with
SEQUEST in Proteome Discover 2.2 (Thermo Fisher Scientific). With
consideration of static modification of carbamidomethylation (C) and
variable modifications of oxidation (O) and deamidation (N), the results
were filtered with peptide rank and peptide confidence (maximum rank, 1;
minimum confidence, high) and protein filters as minimal of 2 peptides per
protein. The FDR rate was set to 1%. MS/MS spectra were extracted with a
tolerance of 10 ppm from raw files.
The cleaved glycans were collected and desalted by NuTip Carbon
(Hypercarb) (Glygen, MD). Glycans were analyzed by a Shimadzu MALDI
(matrix-assisted laser desorption/ionization) mass spectrometer. Glycan
composition was determined by GlycoWorkbench data analysis
software27 (link) and
verified by MS/MS if necessary.
analysis software, GPQuest 2.03 (link),26 (link) Any
peptide-spectrum match (PSM) without glycan annotation and a glycan
intensity ratio less than 20% were filtered out with consideration of a
false discovery rate (FDR) less than 1%.
Deglycosylated peptides and global peptides were searched with
SEQUEST in Proteome Discover 2.2 (Thermo Fisher Scientific). With
consideration of static modification of carbamidomethylation (C) and
variable modifications of oxidation (O) and deamidation (N), the results
were filtered with peptide rank and peptide confidence (maximum rank, 1;
minimum confidence, high) and protein filters as minimal of 2 peptides per
protein. The FDR rate was set to 1%. MS/MS spectra were extracted with a
tolerance of 10 ppm from raw files.
The cleaved glycans were collected and desalted by NuTip Carbon
(Hypercarb) (Glygen, MD). Glycans were analyzed by a Shimadzu MALDI
(matrix-assisted laser desorption/ionization) mass spectrometer. Glycan
composition was determined by GlycoWorkbench data analysis
software27 (link) and
verified by MS/MS if necessary.
