Title : Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry
Abstract :
- The enormous complexity, wide dynamic range of relative protein abundances of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenge the capabilities of existing analytical methodologies
- Here, we describe an approach for broad analysis of human plasma N-glycoproteins using a combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity
- Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column
- N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin and enzymatically digested, and the bound N-linked glycopeptides were released using peptide-N-glycosidase F ( PNGase F)
- Following strong cation exchange ( SCX ) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)
- Using stringent criteria, a total of 2053 different N-glycopeptides were confidently identified, covering 303 nonredundant N-glycoproteins
- This enrichment strategy significantly improved detection and enabled identification of a number of low-abundance proteins , exemplified by interleukin-1 receptor antagonist protein (approximately 200 pg/mL), cathepsin L (approximately 1 ng/mL), and transforming growth factor beta 1 (approximately 2 ng/mL)
- A total of 639 N-glycosylation sites were identified, and the overall high accuracy of these glycosylation site assignments as assessed by accurate mass measurement using high-resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) is initially demonstrated
Output (sent_index, trigger,
protein,
sugar,
site):
- 2. N-glycoproteins, , N-glycoproteins, -, -
- 2. glycoprotein, , glycoprotein, -, -
- 4. glycopeptides, , -, -, glycopeptides
- 4. glycoproteins, , glycoproteins, -, -
- 5. deglycosylated, , -, -, peptides
- 6. N-glycopeptides, , -, -, N-glycopeptides
- 6. N-glycoproteins, , N-glycoproteins, -, -
- 8. N-glycosylation, , -, -, sites
- 8. glycosylation, , -, -, site
Output(Part-Of) (sent_index,
protein,
site):
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):