Supplemental Experimental Procedures
Cell Culture
Chang Liver cells were cultured at 37℃ in 5% CO2 in RPMI 1640 medium (Gibco, USA)
supplemented with 10% newborn calf serum, 100 U/mL penicillin and 100μg/mL streptomycin
(Gibco, USA). HepG2 cells, derived from human hepatocellular carcinoma, were cultured at 37℃
in 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, USA) supplemented with
10% fetal bovine serum, 100 U/mL penicillin and 100μg/mL streptomycin.
Periodate Oxidation
Chang Liver cells and HepG2 cells were all grown to ~90% confluence. The media were
aspirated from each plate of cells and the cells were rinsed twice with 5 mL of PBS each time.
Cells were oxidized with 3 mL 5 mM NaIO4 in oxidation buffer (100 mM NaAc, 150 mM NaCl,
pH 5.5) in the dark at 25℃ for 1 h with gentle shaking, and then the periodate solution was
removed by aspiration. The cells were harvested using a cell scraper (Corning, USA). Cells were
pelleted and rinsed twice with 5 mL of oxidation buffer each time.
Cell Lysis
Cell pellets were homogenized in ice-cold 50 mM HEPES (pH 7.5), 65 mM dithiothreitol
(DTT), 1 mM PMSF, 1% Triton and 2% cocktail (Sigma-Aldrich, USA) using an ultrasonic cell
disrupter (Power was set at 400 W and time of ultrasonicaton was set at 3 s with 3 s intervals for
180 times). Homogenized lysates was centrifuged at 13000g at 4℃for 60 min and the supernatant
was carefully collected. The protein concentration of the supernatant was determined by Bradford
assay.
N-linked Glycoprotein Enrichment by Hydrazide Chemistry
The N-linked glycoproteins in each cell lysates were captured with hydrazide chemistry as
described in a published protocol with minor modification[1]. 100 μL Bio-Rad Affi-Gel Hz
hydrazide resin (bed volumn) was washed with coupling buffer (100 mM NaAc, 150 mM NaCl,
pH 5.5) twice. About 1 mg of extracted proteins from both Chang liver cells and HepG2 cells were
added to prewashed hydrazide resin. The coupling reaction was carried out overnight at room
temperature with adequate shaking. The resin was washed with 1.5 M NaCl, methanol and 100
mM NH4HCO3 sequentially to remove non-specifically bound proteins.
Release of Non-glycopeptides and N-glycopeptides
After removal of NH4HCO3 solution from the last wash, the resin with captured glycoproteins
was diluted with 8 M urea/100 mM NH4HCO3. The glycoproteins on the resin were reduced in 20
mM DTT at 37℃ for 2 h and carboxyamidomethylated in 40 mM iodoacetamide at room
temperature for 40 min in the dark. The supernatant was removed and 100 mM triethyl ammonium
bicarbonate (TEAB) (Sigma-Aldrich, USA) was added to the resin. Trypsin was added with a
weight ratio of trypsin (Sigma-Aldrich, USA) to protein at 1/25 and incubated at 37℃overnight.
The tryptic peptides were carefully collected by gentle centrifugation. The resin was further
washed with 100 mM TEAB buffer. The supernatant was collected and combined with the tryptic
peptides. The N-linked glycopeptides were released by adding 500 unit PNGase F (New England
Biolabs, USA) in 100mM NH4HCO3 to the resin and incubating at 37℃overnight. The released
N-glycopeptides were carefully collected by gentle centrifugation.
Isotopic labeling of the Released Non-glycopeptides
Non-glycopeptides from Chang Liver and HepG2 cells were labeled with light and heavy
dimethyl respectively by an in-solution stable isotope dimethyl labeling approach according to the
published protocol[2]. The separately labeled samples were mix together and desalted with a
homemade C18 solid phase extraction column. After dried down in a Speed Vac (Thermo, San
Jose, CA, USA), the sample was reconstituted in 0.1% FA aqueous solution for 2D nano
LC-MS/MS analysis.
Isotopic labeling of Both Non-glycopeptides and N-glycopeptides
All the procedures before releasing N-glycopeptides were the same as the procedures described
above. Then N-linked glycopeptides were released by adding 1μL of PNGase F (500 unit per μL)
in 100 μL G7 buffer (50 mM sodium phosphate, pH=7.5) (New England Biolabs, USA) to the
resin and incubating at 37℃ overnight. The released N-glycopeptides were carefully collected by
gentle centrifugation. The resin was further washed with 100 μL G7 buffer. The supernatant was
collected and combined with the N-glycopeptides.
Both non-glycopeptides and N-glycopeptides from Chang Liver and HepG2 cells were labeled
with light and heavy dimethyl respectively by an in-solution stable isotope dimethyl labeling
approach according to the published protocol. The separately labeled samples were mix together
and desalted with a homemade C18 solid phase extraction column. After dried down in a Speed
Vac, the non-glycopeptides mixture was reconstituted in 0.1% FA aqueous solution for 2D nano
LC-MS/MS analysis while the released N-glycopeptides mixture was for 1D nano LC-MS/MS
analysis.
Control Experiment: Differential Analysis of Cell Surface Glycoproteins between two
HepG2 cells
The control experiment was carried out to analyze the expression difference of cell surface
glycoproteins between the two HepG2 cells. The procedures of control experiment are the same as
the procedures described above, with enrichment for N-linked glycoprotein by hydrazide
chemistry,
glycopeptides
for
identification
of glycoproteins
and
isotopically labeled
non-glycopeptides for quantification of glycoproteins, except that the starting cell lines is the same
cell line, i.e. HepG2 cells, not two different cell lines.
N-linked Glycopeptide Enrichment by Hydrazide Chemistry
About 1 mg of extracted proteins from both Chang Liver cells and HepG2 cells were first
reduced in 20 mM DTT at 60℃ for 1 h and carboxyamidomethylated in 40 mM iodoacetamide at
room temperature for 40 min in the dark. Then trypsin was added with a weight ratio of trypsin to
protein at 1/25 and incubated at 37℃ overnight.
The tryptic peptides were desalted with a homemade C18 solid phase extraction column. After
dried down in a Speed Vac, the sample was reconstituted in 200ul coupling buffer (100 mM
sodium acetate, 150 mM NaCl, pH 5.5) for glycopeptides enrichment. 50ul prewashed Bio-Rad
Affi-Gel Hz hydrazide resin (bed volumn) was used for coupling reaction and the coupling
reaction was carried out overnight at room temperature with adequate shaking. The resin was
washed with 1 mL of 1.5 M NaCl, methanol and 100 mM NH4HCO3 sequentially three times to
remove nonspecifically bound proteins. After removal of NH4HCO3 solution from the last wash,
1μL of PNGase F (500 unit per μL) in 200μL G7 buffer was added to the resin and incubated at
37℃ overnight. The supernatant was collected and labeled with light and heavy dimethyl
respectively by an in-solution stable isotope dimethyl labeling approach according to the
published protocol. The labeled deglycosylated peptides mixture was for 1D nano LC-MS/MS
analysis.
LC-MS/MS Analysis
For each biological replicate (n=2), three technical replicates were analyzed by LC-MS/MS.
The non-glycopeptides was analyzed by a 2D nano LC-MS/MS system as described previously[3]
with minor change. The strong cation exchange (SCX) column was connected to a RP C18 column
(12 cm × 75μm i.d) in tandem by a union. The SCX column was prepared by the same method as
described by Wang et al.[4]. For the C18 capillary column, one end of the fused-silica capillary
was manually pulled to a fine point of ~5μm with a flame torch, and then the C18 AQ beads (5μm,
12 nm) from Microm BioResources (Auburn, CA, USA) were packed until the packing section
reached a length of 12 cm using a pneumatic pump. The RP C18 column was directly coupled
with a LTQ-Orbitrap XL mass spectrometry (Thermo Finnigan, San Jose, CA, USA) with a
nanospray source.
The RPLC-MS/MS was performed on a nano-RPLC-MS/MS system. A Finnigan surveyor MS
pump (Thermo Finnigan, San Jose, CA, USA) was used to deliver mobile phase. 0.1% Formic
acid water solution (buffer A) and ACN with 0.1% formic acid (buffer B) were used for the
generation of linear gradient for RPLC separation. 1000 mM NH4Ac (pH 2.7) as buffer C was
used to generate step salt gradients to gradually elute the non-glycopeptides which were captured
on SCX column onto the RP C18 analytical column after 25μL reconstituted non-glycopeptides
were loaded automatically onto a phosphate SCX monolithic column (7 cm × 150μm i.d.). The
serial stepwise salt concentrations of NH4Ac used for 10 step salt gradients were 50, 100, 150, 200,
250, 300, 350, 400, 500 and 1000 mM, respectively. Each salt step lasted 10 min and then the
system was re-equilibrated with buffer A for another 15 min. After each salt elution, binary
separation gradient from 5% to 35% in 100 min with a flow rate of ∼300 nL/min was started for
reversed phase separation.
The MS analysis was performed on LTQ-Orbitrap XL mass spectrometer at a resolution of 60
000. The temperature of the ion transfer capillary was set at 200℃. The spray voltage was set at 1.8
kV and normalized collision energy was set at 35%. One microscan was set for each MS and
MS/MS scan. All MS and MS/MS spectra were acquired in the data-dependent mode. The mass
spectrometer was set that one full MS scan was followed by six MS/MS scans on the six most
intense ions by CID. The scan range was set from m/z 400 to m/z 2000. The target ion setting was
5e5, with a maximum fill-time of 500 ms. Minimum signal threshold set for MS/MS scan was 500.
The dynamic exclusion function was set as follows: repeat count 2, repeat duration 30 s, and
exclusion duration 60 s.
The released N-glycopeptides were analyzed by 1D nano LC-MS/MS, in which the
glycopeptides sample was loaded directly onto a RP C18 column and 120min binary separation
gradient from 5% to 35% with a flow rate of ∼300 nL/min was used for separation. The MS
analysis was performed as described above.
Protein Identification and Quantification
The process of the datasets of 2D nano LC-MS/MS was the same as described previously[3].
DTASupercharge (v2.0a7)[5] was applied to convert all the MS/MS spectra in one acquired raw
file to a single *.mgf file. The *.mgf file was searched against a composite database including
original and reversed human protein database of International Protein Index (ipi.human 3.17 fasta,
including 60 234 entries, http://www.ebi.ac.uk/IPI/IPIhuman.html) by MASCOT Version 2.1
(Matrix Science, UK). Carboxamidomethylation (+57.0215 Da) of Cysteine residues was set as
static modification and oxidation of methionine residues was searched as variable modification of
+15.9949 Da. Also both light and heavy dimethylation of peptide N termini and the side chain of
lysine residues were set as variable modification of +28.0313 Da and +32.0564 Da, respectively.
Peptides were searched using fully tryptic cleavage constraints and a maximum of two missed
cleavages sites were allowed for tryptic digestion. 10 ppm was set for the mass tolerances of
parent masses and 0.8 Da for fragment masses. Peptides with Mascot score ≥25 (rank 1, P ≤0.05)
were used for protein quantification. The false detection rate (FDR) was determined by equation
of FDR=[2* FP/(FP+TP)]*100, where FP (false positive) is the number of peptides that were
identified based on sequences in the reverse database component and TP (true positive) is the
number of peptides that were identified based on sequences in the forward database component.
FDR of each 2D nano LC-MS/MS analysis was controlled to be less than 1% by setting different
Mascot score.
A dimethyl-adapted version of MSQuant (v2.0a81) was applied for protein quantification.
Peptide ratios were obtained by calculating the peak area of extracted ion chromatograms (XIC) of
the light and heavy forms of the peptide using the monoisotopic peaks only and protein ratios were
calculated from the average of the all quantified peptides[5, 6]. Then all the MSQuant outputs of
all SCX fraction were imported into StatQuant (v1.2.2) and the quantified proteins were
normalized against the log2 of the median ratio of all peptides quantified [6, 7].
Deamidation of both glutamines and asparagines (+0.9840 Da) were added as another variable
modifications in the process of analyzing the results of 1D nano LC-MS/MS by MASCOT. The
search result was exported in csv format and the Trans-Proteomics Pipeline (TPP, v4.3.1)[8] was
used to convert raw file to ‘mzxml’ format. An in-house developed software Armone v1.0 beta 2[9]
was applied to process the *.csv file together with *.mzxml file. FDR of the final identifications
was controlled less than 1% by setting different filtering criterion. The proteins identified by the
same peptides were grouped. To remove the redundancy of identifications, only one protein was
kept for each group by using the following order as described by He et al.[10] : keep the proteins
with annotation in Swiss-Prot and RefSeq_NP, keep the proteins with annotation in Swiss-Prot,
and then keep the proteins with annotation in RefSeq_NP. If still over two proteins remained, the
protein with longest sequence was kept as the unique entry in the group.
Transmembrane domains were predicted by the common topology algorithm TMHMM v2.0, a
hidden Markov model-based predictor for transmembrane helices in protein sequences[11].
Subcellular location analysis was performed using GoMiner[12] with the new database (version
go_201202).
Western Blotting
Equal amount of extracted proteins from both Chang Liver cells and HepG2 cells were
separated on a 12% SDS-PAGE gel and transferred to nitrocellulose membrane. Antibodies
against basigin (EMMPRIN) and basal cell adhension molecule (BCAM) (Santa Cruz
Biotechnology, CA, USA) were used to detect the protein of interest. Both primary antibodies
were used with a dilution of 1:1000 and a second antibody against rabbit IgG (Santa Cruz
Biotechnology, USA, 1:5000 dilution) was used for both proteins. Blots were developed with ECL
western blotting reagent (Pierce, IL, USA). For western blot loading controls, β-actin monoclonal
antibody (Cell Signaling Technology, USA) was used.
Supplemental Figures
Figure S1
(A)
(B)
(C)
(D)
(E)
(F)
Venn diagrams showing the overlapping and unique data of three runs of analyses.
(A) 193 glycoproteins (RSD=2.1%, n=3) were identified in each run of three replicate runs of 1D
LC-MS/MS in first biological replicate and more than 65% of total identified proteins in three
runs can be identified in two or three runs.
(B) 208 glycoproteins (RSD=5.5%, n=3) were identified in each run of three replicate runs of 1D
LC-MS/MS in another biological replicate and more than 70% of total identified proteins in
three runs can be identified in two or three runs.
(C) 458 unique glycopeptides (RSD=2.4%, n=3) were identified in each run of three replicate runs
of 1D LC-MS/MS in first biological replicate.
(D) 508 unique glycopeptides (RSD=5.7%, n=3) were identified in each run of three replicate runs
of 1D LC-MS/MS in another biological replicate.
(E) 5379 unique tryptic peptides (RSD=10.4%, n=3) were identified in each run of three replicate
runs of 2D LC-MS/MS in first biological replicate.
(F) 5084 unique tryptic peptides (RSD=14.9%, n=3) were identified in each run of three replicate
runs of 2D LC-MS/MS in another biological replicate.
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