Isolation of Nlinked glycopeptides from plasma

1
Isolation of N-linked glycopeptides from plasma
Yong Zhou1, Ruedi Aebersold2, and Hui Zhang1,3
1Institute for Systems Biology, Seattle,
Washington 98103, USA 2Institute of Molecular
Systems Biology, Swiss Federal Institute of
Technology (ETH) Zurich and Faculty of Natural
Sciences, University of Zurich, CH-8093,
Switzerland 3Department of Pathology, Johns
Hopkins Medical Institutes, Baltimore, Maryland
21287, USA
OVERVIEW
RESULTS
1. Monitoring each step of SPEG procedure using
mouse plasma spiked with 14C-labeled human
blood glycoprotein 14C-Radioactivity assay
offers very similar pattern of coupling
efficiency comparing with N-linked glycopeptides
quantified with LC-MS/MS and isotopic labeling.
Here, the coupling efficiency for different NaIO4
oxidation conditions were determined by either
direct 14C-radioactivity assay of uncoupled
fractions post coupling (A) or captured N-linked
glycopeptides quantified by LC-MS/MS and
isotopic-labeling (B). (C), The CID spectrum of
one peptide from spiked human AG with specific
M/Z value (2021.8498) and sequence
(QDQCIYNTTIYNVQR). (D), The ASAPRatio of the
peptide identified in (C)its abundance in Light
(1mM NaIO4) is only 0.209?0.013 fold of Heavy
(10mM NaIO4), i.e. one fifth.
Optimizing the recently developed Solid-Phase
Extraction of (N-linked) Glycopeptides (SPEG)
procedure for blood plasma analysis by spiking in
14C-labeled human glycoproteins significantly
enhanced the specificity and yield of the
isolated N-linked glycopeptides. This will
improve the sensitivity of discovery of clinical
biomarkers from blood.
3. Optimizing each step
4. A flow chart illustrating the optimized
peptide-level SPEG procedure
1) Trypsin Digestion
Here, five conditions were tested, including
100 mM NH4HCO3 8M Urea in 100 mM
NH4HCO3 (original) 8M Urea in 100 mM
NH4HCO3, plus 2 mM CaCl2 0.1 RapiGest
in 100 mM NH4HCO3 and 50 TFE
(Triflouroethonal) in 100 mM NH4HCO3.
INTROCUCTION
There is growing interest in discovery of disease
biomarkers from blood plasma. For this reason,
quantitative analysis of plasma proteins has been
the focus of different proteomic technologies.
The challenges faced by all quantitative plasma
proteomic methods include complexity and the high
dynamic range of the plasma sample. Therefore, a
desirable proteomic technique for plasma
profiling must be sensitive, reproducible, and
robust. Recently, we have developed a method for
Solid-Phase Extraction of N-linked Glycopeptides
(SPEG), and we have shown that analysis of plasma
using SPEG improves dynamic range and
sensitivity. Here, we optimized each step of the
method and developed a standard procedure for
plasma analysis using SPEG and mass spectrometry
by spiking in two 14C-labeled human
glycoproteins.
A), Released 14C radioactive activities from
beads by tryptic digestion B), PNGase F released
14C radioactive activities C), PNGase F
unreleased 14C radioactive activities.
METHODS
2) Optimization of PNGase F release and
peptide-level SPEG flow by 14C radioactivity
monitoring A), Yield comparison PNGase F
released radioactivity from spiked 14C-AG
between protein-level and peptide-level SPEG B),
Saturation curve of hydrazide resin for
glycocapture of digested glycopeptides from 20 ?l
plasma spiked with 14C-AG. Data shown as
percentage of original radioactivity retained on
hydrazide resin after overnight coupling C),
Yield of N-linked glycopeptides via peptide-level
SPEG by different volume of Affi-gel hydrazide
slurry D), Yield of N-linked glycopeptides via
peptide-level SPEG by different amount of PNGase
F-- 1.5 ?l PNGase F (1,250 U, New England Biolabs
unit) is enough for 20 ?l mouse plasma.
CONCLUSION
Here weve described the optimization of a
recently developed Solid-Phase Extraction of
(N-linked) Glycopeptides (SPEG) procedure for
blood plasma analysis by spiking with
14C-labeled human glycoproteins, which
significantly enhances the specificity and yield
of the isolated N-linked glycopeptides. After the
optimization of each step of the SPEG procedure
as discussed above, one new flow of N-linked
glycopeptide capture can be drawn out as in
Figure 4. The experiments show that 25 µl of
hydrazide resin, 10mM NaIO4 for 1 hour at room
temperature, and 1.5 µl PNGase F for 14 hours at
37 C yield optimal recovery of N-linked
glycopeptides from tryptic peptides coming from
20 µl of mouse blood plasma. The buffer system
for trypsin digestion is a critical factor for
SPEG specificity and efficiency, with 50 TFE
giving the best results. Finally, pre-digestion
of plasma proteins using trypsin was shown to be
one effective way to significantly increase the
yield of SPEG. These results will improve the
sensitivity of discovery of clinical biomarkers
from blood.
14C-IODOACTAMIDE LABELING HUMAN GLYCOPROTEINS
2. Evaluating the performance of the original
SPEG procedure using mouse plasma spiked with
14C-labeled two human blood glycoproteinsAG
and AT
?-1-acid glycoprotein 1 (AG) (Positive Control 2
Cys in 2 Cys out) MALSWVLTVLSLLPLLEAQIPLCANLVPVPI
TNATLDQITGKWFYIASAFRNEEYNKSVQEIQATFFYFTPNKTEDTIFL
REYQTRQDQCIYNTTYLNVQRENGTISRYVGGQEHFAHLLILRDTKTYM
LAFDVNDEKNWGLSVYADKPETTKEQLGEFYEALDCLRIPKSDVVYTDWK
KDKCEPLEKQHEKERKQEEGES ?-1-antitrypsin (AT)
(Negative Control 0 Cys in 1 Cys
out) MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHP
TFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKA
DTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLF
LSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGK
IVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVK
VPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENEL
THDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGA
DLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVK
FNKPFVFLMIEQNTKSPLFMGKVVNPTQK
REFERENCES
1 Hui Zhang, Xiao-jun Li, Daniel Martin and
Ruedi Aebersold, Identification and
quantification of N-linked glycoproteins using
hydrazide chemistry, stable isotope labeling and
mass spectrometry. Nature Biotechnology. 2003,
21660-666.