Supporting Information
Designed
Synthesis
Mesoporous
of
Microspheres
Fluorous-Functionalized
for
Specific
Magnetic
Enrichment
of
Phosphopeptides with Fluorous Derivatization
Experimental section
1. Materials and reagents.
Iron (Ⅲ) chloride hexahydrate (FeCl3·6H2O), ethylene glycol, ethanol, sodium acetate
anhydrous (NaAc) and sodium hydroxide (NaOH) were purchased from Sinopharm Chemical
Reagent Co., Ltd. (Shanghai, China). Hexadecyltrimethylammonium bromide was purchased
from Sigma Chemical (St. Louis, MO, USA). Tetraethylsilicate (TEOS) was purchased from
Shanghai
Lingfeng
Chemical
Reagent
Co.,
Ltd.
(Shanghai,
China).
1H,1H,2H,2H-perfluorodecyltriethoxysilane was purchased from Alfa Aesar (Tianjin, China).
The NdFeB magnet was purchased from PCCW Ltd. (Beijing, China), 2 cm long, 2 cm wide,
1 cm high, with surface magnetic field strength of 1000 Gauss.
Ammonium bicarbonate (NH4HCO3) was purchased from Chinasun Specialty Products Co.,
Ltd (Jiangsu, China). L-1-tosylamido-2-phenylethylchloromethyl ketone (TPCK) treated
trypsin (from bovine pancreas), PNGase F (Genetimes Technology), bovine β-casein from
bovine milk (95% PAGE), bovine serum albumin (BSA), chicken egg ovalbumin (OVA),
1H,1H,2H,2H-perfluorooctane-1-thiol, 2,5-dihydroxybenzoic acid (DHB) and trifluoroacetic
acid
(TFA)
were
purchased
from
Sigma
Chemical
(St.
Louis,
MO,
USA).
1H,1H-perfluorooctylamine (PFOA) was purchased from J&K Chemical (Shanghai, China).
The standard phosphorylated peptide (SADGQHAGGLVK, Mr = 1219.24 Da) was purchased
from ChinaPeptides Co., Ltd. (Shanghai, China). Acetonitrile (ACN) was purchased from
Merck (Darmstadt, Germany). Dimethyl sulfoxide (DMSO) and hydrogen peroxide (30%,
w/w) were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China).
1
Barium hydroxide (Ba(OH)2) was purchased from Sinopharm Chemical Reagent Co., Ltd.
(Shanghai, China). The human serum sample originated from a hepatocellular carcinoma
patient was acquired from Shanghai Zhongshan Hospital. Deionized water was purified by a
Milli-Q system (Milford, MA, USA).
All other chemicals and reagents are of the highest grade commercially available and used
as received.
2. Synthesis of Fe3O4 microspheres
The Fe3O4 microspheres were prepared via a hydrothermal reaction frequently used by our
groups [1]. Briefly, 1.35 g of FeCl3·6H2O was dissolved in 75 mL of ethylene glycol under
magnetic stirring. After complete dissolution, 3.60 g of NaAc was added. After the mixture
was adequately stirred for another 1 h, the resulting solution was sealed in a Teflon-lined
stainless-steel autoclave and heated at 200 ℃ for 16 h. The Fe3O4 microspheres obtained were
washed with deionized water and ethanol several times in turn.
3. Characterizations and measurements
Transmission electron microscope (TEM) images were taken on a JEM-2011 electron
microscope (JEOL, Japan) operated at 200 kV. The sample was dispersed in ethanol in
advance and collected for analysis by using carbon film-covered copper grids. Scanning
electron microscope (SEM) images and the energy dispersive X-ray (EDX) spectrum were
recorded on a Phenom Prox electron microscope (Phenom, Netherlands) operating at 15 kV.
The sample was sprayed by a thin palladium film before the observation. Fourier transform
infrared (FT-IR) spectra were collected on a Nexus 470 Fourier spectrophotometer (Nicolet,
USA) using KBr pellets. Nitrogen adsorption-desorption isotherms were measured at 77 K
with a Micromeritcs Tristar 3000 analyzer (Tristar, USA). The Brunauer-Emmett-Teller (BET)
method was utilized to calculate the surface surface area and the Barrett-Joyner-Halenda (BJH)
method was used to estimate the average pore size. The magnetic characterization was
performed on a MPMS (SQUID) Vibrating Sample Magnetometer (Quantum Design, USA).
4. Sample preparation
2
Bovine β-casein and BSA proteins were dissolved in 25 mM NH4HCO3 buffer (pH 8.3)
respectively and digested with trypsin (2.5%, w/w) at 37 ℃ for 16 h. The tryptic digests could
be diluted to lower concentrations with 25 mM NH4HCO3 solution.
To prepare the DHB matrix solution, we dissolved 20 mg of 2,5-dihydroxybenzoic acid in 1
mL of 50% ACN (v/v) aqueous solution containing H3PO4 (1%, v/v).
Before investigating the enrichment efficiency of Fe3O4@mSiO2-C8F17 for fluorous
labelled peptides, we suspended the magnetic microspheres in deionized water to prepare a
dispersion (10 mg/mL). After vibration with the help of ultrasonication, a clear and
homogeneous solution was obtained. Under a magnetic field, the Fe3O4@mSiO2-C8F17
microspheres were immediately attracted from the solution to the sidewall of the vial within 2
s, which saved much time for the enrichment procedures.
5. Enrichment of fluorous derivatized phosphopeptides
To enrich fluorous derivatized phosphopeptides from the β-casein tryptic digest, 20 μL of
Fe3O4@mSiO2-C8F17 suspension was added to 200 μL of the derivatized digest (2 μL of
fluorous derivatized β-casein tryptic digest spiked into 198 μL of deionized water) in a 0.6 mL
centrifuge tube. Then, the mixture was vibrated in a vortex at 37 ℃ for 30 min. The magnetic
microspheres were isolated from the supernatant under a magnetic field. Next, the magnetic
microspheres were rinsed with deionized water three times and eluted with 10 μL of
acetonitrile at 37 ℃ for 15 min. The eluent was deposited on a MALDI sample target
(Applied Biosystems/MDS SCIEX, Foster City, CA, USA) and dried at room temperature.
The dilution without enrichment and the supernatant of equal volume were also deposited on
the same sample target for comparison. Later on, 0.8 μL of DHB matrix was pipetted on the
sample spots. Four replicate spots were taken for every sample. After the evaporation of the
solvent, the samples were submitted to MALDI-TOF MS for identification.
For the fluorous derivatized mixtures of β-casein tryptic digest and BSA tryptic digest, and
for the fluorous derivatized human serum, similar dilution, enrichment, washing, elution and
spotting steps were followed. The adducts of samples and DHB matrix were analyzed by
MALDI-TOF MS. However, after the fluorous labelled mixtures of β-casein tryptic digest and
BSA tryptic digest were enriched by Fe3O4@mSiO2-C8F17, the adsorbents were washed with
3
0.2 M ammonium to remove the interfering non-fluorous peptides.
6. Fluorous derivatization of glycans
An OVA solution at the concentration of 1 μg/μL was treated with PNGase F to release the
glycans, and the enzymatic deglycosylation reaction was conducted at 37 ℃ for 18 h. The
resultant glycans were dissolved in 50% ACN/5% acetic acid (v/v) buffer and were allowed to
react with 1H,1H-perfluorooctylamine (the molar ratio of derivatization reagent to glycans
was 1:10) at 65 ℃ for 2h, with NaBH3CN as the reducing reagent.
4
Scheme S1. The synthetic route to Fe3O4@mSiO2-C8F17 microspheres.
5
Figure S1. (a) The SEM image of the spot chosen for the energy dispersive X-ray (EDX)
analysis and (b) The EDX spectrum of the Fe3O4@mSiO2-C8F17 microspheres.
6
Figure S2. The magnetization curve of Fe3O4@mSiO2-C8F17.
7
8
Figure S3. MALDI-TOF mass spectrum for the dilution of fluorous derivatized β-casein
tryptic digest after treatment with Fe3O4@mSiO2-C8F17 (a); and the corresponding
MALDI-TOF/TOF tandem mass spectra: precursor ion m/z 2343.0 (b), precursor ion m/z
2838.0 (c) and precursor ion m/z 3404.0 (d). Fluorous phosphopeptides identified are marked
with capital F.
9
Figure S4. MALDI-TOF mass spectra for the dilution of fluorous derivatized standard
phosphopeptide. The original phosphopeptide is marked with capital P and the fluorous
derivatized phosphopeptide is marked with capital F.
10
Figure S5. MALDI-TOF mass spectra for the N-glycans derived from fluorous derivatized
ovalbumin digests (1 μg/μL): (a) before and (b) after enrichment with Fe3O4@mSiO2-C8F17.
[M + H]+ signals are denoted as capital F and [M + Na]+ signals are denoted as capital Na.
11
Figure S6. MALDI-TOF mass spectra for the dilution of fluorous derivatized human
serum: (a) before and (b) after enrichment with Fe3O4@mSiO2-C8F17. Fluorous
derivatized phosphopeptides identified are marked with capital F.
12
Table S1. The EDX analysis results of Fe3O4@mSiO2-C8F17
Element Symbol
Element Name
Weight Concentration
Error
O
Oxygen
38.7
0.0
Fe
Iron
32.6
0.2
F
Fluorine
23.9
0.4
Si
Silicon
4.0
0.1
C
Carbon
0.8
3.6
Table S2. Detailed information for the fluorous derivatized phosphopeptides
identified from the fluorous derivatized β-casein tryptic digests after enrichment with
Fe3O4@mSiO2-C8F17
Amino acid sequence of
Observed
Mr (expt)
Mr (calc)
derivatized phosphopeptides
RELEELNVPGEIVECLSS
3403.3557
3404.5331
3404.3246
SEESITR+F13-Alk(C)
FQCEEQQQTEDELQDKI
2836.9656
2838.3280
2838.1713
HPF+F13-Alk(C)
FQCEEQQQTEDELQDK+
2342.9160
2344.0408
2343.9301
F13-Alk(C)
2209.3047
2210.2879
2209.3993a
DIGCESTEDQAMETIK+F
13-Alk(C)
2115.4172
2115.1776
2114.27396a
YLGEYLIVPNCAEER+F1
3-Alk(C)
1942.0817
1942.8778
1942.8459a
VPQLEIVPNCAEER+F13Alk(C)
1843.2764
1844.2309
1844.1891a
EQLCTSEENSKK+F13-Al
k(C)
1748.2822
1748.7685
1748.7320a
TVDMECTEVFTK+F13-A
lk(C)
13
FQCEEQQQTEDELQDKI
1562.2347
1561.1010
1561.0187
HPF+F13-Alk(C)
a The phosphopeptides derived from α-casein.
Table S3. Detailed information of derivatized N-glycans released from OVA
glycoproteins enriched by Fe3O4@mSiO2-C8F17 (N-linked glycans were released by
PNGase F digestion, and glycan structures were searched with Glycoworkbench
through the molecular weight of oligosaccharide.) ■
(blue)
represents
N-acetylglucosamine, ● (green) represents mannose, ● (yellow) represents
galactose, and PFOA represents 1H,1H-perfluorooctylamine.
Observed
Mr (expt)
Mr (calc)
1315.49
1316.28
1316.47
1495.52
1496.39
1496.43
1618.55
1617.47
1617.55
1659.59
1658.39
1658.46
1721.65
1722.45
1722.57
1801.57
1802.48
1802.58
1842.67
1843.52
1843.69
Structure
14
1903.83
1902.59
1902.65
2065.76
2064.61
2064.71
2127.78
2128.58
2128.60
2248.84
2249.70
2249.83
2266.70
2267.67
2267.79
2307.82
2308.79
2308.89
2412.82
2411.78
2411.88
2471.88
2470.68
2470.89
2535.84
2534.74
2534.88
2654.89
2655.75
2655.85
15
Table S4. Detailed information for the fluorous phosphopeptides identified from the
fluorous
derivatized
human
serum
samples
after
enrichment
with
Fe3O4@mSiO2-C8F17
m/z of fluorous derivatized
m/z of corresponding
phosphopeptides
phosphopeptides
1671
1389
D[pS]GEGDFLAEGGGV
1742
1460
AD[pS]GEGDFLAEGGGV
1827
1545
D[pS]GEGDFLAEGGGVR
1898
1616
AD[pS]GEGDFLAEGGGVR
1489
1206
RG[pS]FSSENTWRK
1591
1309
K[pS]LPVSPVWGFKE
Amino acid sequence
Reference
[1] Xu, X., Deng, C., Gao, M., Yu, W., Yang, P., Zhang, X., Synthesis of magnetic
microspheres with immobilized metal ions for enrichment and direct determination of
phosphopeptides by matrix-assisted laser desorption ionization mass spectrometry.
Adv. Mater. 2006, 18, 3289-3293.
16