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
1. Monitoring each step of SPEG procedure using
mouse plasma spiked with [14C]-labeled human
blood glycoprotein
PNGase F cleavage
from beads
H2N
COOH
Released N-linked
glycopeptides
H2N
H2N
COOH
Washing
H2N
Relative Glycopeptide
Abundance in MS
#
H2N
COOH
H2N
COOH
H2N
Tryptic peptides on
hydrazide beads
COOH H2N
H2N
0.75
0.50
**
14C-IODOACTAMIDE
LABELING HUMAN
Oxidized glycans
-1-acid glycoprotein 1 (AG)
(Positive Control: 2 Cys in; 2 Cys out)
MALSWVLTVLSLLPLLEAQIPLCANLVPVPITNATLDQITGKWFYIASAFRNEEYNKSVQ
EIQATFFYFTPNKTEDTIFLREYQTRQDQCIYNTTYLNVQRENGTISRYVGGQEHFAHLL
ILRDTKTYMLAFDVNDEKNWGLSVYADKPETTKEQLGEFYEALDCLRIPKSDVVYTDWKK
DKCEPLEKQHEKERKQEEGES
-1-antitrypsin (AT)
(Negative Control: 0 Cys in; 1 Cys out)
MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFS
LYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGF
QELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQ
INDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTV
KVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFL
ENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKA
VLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK
C18 column
Urea
Urea +CaCl2
RapiGest
0
No Enzy me
TFE
Urea
Urea +CaCl2 RapiGest
10 mM, RT 1h in dark
C18 column clean up
Hydrazide resin coupling
75
25 l Affi-gel slurry
AT
AG
Washing
50
1.5M NaCl, 80% ACN, H2O, 100 mM NH4HCO3
PNGase F release
25
0
No Enzy me
TFE
NaIO4 oxidation
Trypsin Denature Buffers
Urea
Urea +CaCl2 RapiGest
TFE
LC-MS/MS
Trypsin Denature Buffers
0.00
10.0
50.0
1.0
[NaIO4] in mM
C
1.5 l PNGase F, 37C overnight
**
A), Yield comparison: PNGase F released radioactivity from spiked
[14C]-AG between protein-level and peptide-level SPEG;
D
10.0
50.0
[NaIO4] in mM
3.9e+06
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.
COOH
Glycans
Desalting
COOH
QDQCIYNTTYLNVQR, MH+ 2021.8498
Light vs. Heavy: 0.209  0.013
2. Evaluating the performance of the original SPEG
procedure using mouse plasma spiked with [14C]labeled two human blood glycoproteins—AG and AT
A
60
B
*
50
40
30
20
10
80
60
40
20
0
0
Protein Level
Peptide Level
0
40
80
120
160
Affi-gel Vol. (l)
100
AT
AG
75
50
C
75
50
40
30
20
10
0
40
80
Affi-gel Vol. ( l)
Post-trypsin
PNGaseF Released
PNGase Release
On Bead
50
REFERENCES
25
0
0
0
Here we’ve 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 Nlinked 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.
D
60
25
Coupled
CONCLUSION
**
COOH
COOH
H2N
37C overnight, in 5%TFE, 100 mM NH4HCO3
2) Optimization of PNGase F release and peptide-level
SPEG flow by [14C] radioactivity monitoring
1.00
COOH
Hydrazide beads
GLYCOPROTEINS
1.25
Tryptic
digestion
COOH
20
5
% Original Radioactivity
H2N
40
*
10
Trypsin Denature Buffers
**
1.0
Hydrazide captured
glycoproteins
Oxidized glycans
Mouse plasma
+ [14C]AG/AT
Denature/Trypsin proteolysis
14
H2N
60
C
15
% Yield of Coupled [ C]-AG
H2N
H2N
COOH
**
No Enzyme
0.25
0.00
COOH
B
80
14
COOH
0.50
**
20l (~1.5mg protein)
#
0.75
0.25
A
% Yield of Spiked [ C]-AG
H2N
Oxidation (NaIO4)
COOH
1.00
Plasma
A), Released [14C] radioactive activities from beads by tryptic
digestion;
B), PNGase F released [14C] radioactive activities;
C), PNGase F unreleased [14C] radioactive activities.
0
1.50
RT
4O C
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.
**
B
1.25
4. A flow chart illustrating the optimized peptidelevel SPEG procedure
1) Trypsin Digestion:
% Yield of Spiked [ 14C]-AG
H2N
Capturing to
Hydrazide beads
COOH
A
% Original Radioactivity
COOH
[1]
H2N
(D), The ASAPRatio of the peptide identified in (C)—it’s abundance
in Light (1mM NaIO4) is only 0.2090.013 fold of Heavy (10mM
NaIO4), i.e. one fifth.
14
METHODS
SPEG METHOD
(C), The CID spectrum of one peptide from spiked human AG with
specific M/Z value (2021.8498) and sequence
(QDQCIYN#TTIYNVQR).
Relative Coupled Radioactivity
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.
of Spiked [ C]-AG
INTROCUCTION
[14C]-Radioactivity assay offers very similar pattern of coupling
efficiency comparing with N-linked glycopeptides quantified with LCMS/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).
3. Optimizing each step
% Original Radioactivities
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.
% Original Radioactivities
RESULTS
% Original Radioactivities
OVERVIEW
120
160
0
1
2
3
4
PNGase F Volume ( l/tube)
5
[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,
21:660-666.