Supporting Information
Preparation of protein imprinted materials by hierarchical
imprinting techniques and application in selective
depletion of albumin from human serum
Jinxiang Liu †,‡ , , Qiliang Deng † , Dingyin Tao †,‡ , Kaiguang Yang † , Lihua Zhang
†,*
, Zhen Liang †, Yukui Zhang †
† National Chromatographic R. & A. Center, Key Laboratory of Separation Science
for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of
Sciences, Dalian 116023, China
‡ University of Chinese Academy of Sciences, Beijing 100049, China
*Corresponding Author
Address: 457 Zhongshan Road, Dalian 116023, China
E-mail: lihuazhang@dicp.ac.cn
Tel/Fax: +86-411-84379720
Ⅰ. Experimental section
Gravimetric yield of synthesized polymers and swelling ratio measurement toward imprinted
particles by hierarchical imprinting.
Silica particles, with 300 Å, 500Å and 1000Å pore sizes, were applied as the matrix to fabricate
the imprinted particles by hierarchical imprinting. After the completely removal of the proteins
and the silica particles, the synthesized polymer were obtained. The gravimetric yield of
synthesized polymers were calculated by the ratio of the amount of the synthesized polymer to the
initial amount of silica particles.
The swelling ratio of the imprinted particles by hierarchical imprinting was obtained from the
swelling experiments in water at 40℃. 100mg imprinted particles were dried overnight in a
vacuum desiccator. The imprinted particles were accurately weighed (Mi) and immersed in 2 ml
water in closed bottles for 24 hours. Then, the surfaces were dried with filter paper and the
samples were quickly weighed (Mt is the swollen weight of the particles after equilibrium is
reached). The swelling ratio, Q, in water was calculated using the equation (Polym. Eng. Sci. 2005,
45, 1239-1246.):
Q
Mt  Mi
Mi
Protein digestion
The original serum and albumin depleted serum were diluted to 0.2 mg/mL by
denaturing buffer, containing 8 M urea, and 16 μL of 100 mM DTT was added into 1
mL of diluted serum. The protein solution was incubated at 56 °C for 1 h. Then, 20
μL of 200 mM IAA was added, and the solution was further incubated at 37 °C for 30
min in the dark. Subsequently, the mixture was diluted by 10-fold with 50 mM
ammonium bicarbonate buffer (pH 8.0). Finally, trypsin was added in the
trypsin/protein (w/w) ratio of 1:50, and incubated at 37 °C for 12 h. Before usage,
tryptic digests were desalted with a C18 solid-phase cartridge.
2D nano-SCX-RPLC-MS/MS experiments
The analysis of peptides was performed by 2D nano-SCX-RPLC-MS/MS. Two
microgram peptides were firstly loaded onto an SCX trap column (150 μm i.d. ×2 cm,
packed with UNI MC5SP-500 particles) at a flow rate of 10 μL/min. The salt
concentration in each step was 30, 60, 100, 180, 300, and 1000 mM ammonium
acetate (pH 3.0) respectively, and each step was held for 15 min. Peptides were
sequentially eluted onto a C18 capillary column (75 μm i.d. ×14 cm) packed with C18
silica particles (5 μm, 200 Å). Linear gradient, generated by 2% (v/v) acetonitrile with
0.1% (v/v) formic acid (buffer A) and 98% (v/v) acetonitrile with 0.1% (v/v) formic
acid (buffer B), was applied as follows: 0% B for 10 min, to 10% B in 10 min, to 40%
B in 75 min, to 80% B in 5 min, and kept at 80% B for 15 min.
The LTQ instrument was operated in a positive mode. The heated capillary
temperature and the spray voltage were 180 C and 1.8 kV respectively. The collision
energy for MS/MS scanning was 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 as one full MS scan following with nine MS/MS scans
on the nine most intensive ions. The dynamic exclusion function was set as follows:
repeat count, 1; repeat duration, 30 s; exclusion duration, 180 s. Total ion current
chromatograms and mass spectra ranging from m/z 400 to 2000 were recorded with
Xcalibur software (Version 1.4, Thermo System).
Data analysis
MS/MS analysis was performed using SEQUEST search program incorporated in
BioWorks software (version 3.3.1). The database was ipi.HUMAN.v3.17. fasta, and
reversed sequences were appended to the database for the evaluation of false
discovery rate (FDR). Cysteine residues were searched as static modification of
+57.0215 Da, and methionine/asparagine residues were searched as variable
modifications of +15.9949 Da. Peptides were searched using fully tryptic cleavage
constraints and up to two internal cleavages sites were allowed for tryptic digestion.
The mass tolerances were 2 Da for parent masses and 1 Da for fragment masses. The
BuildSummary software was used to reduce the apparent redundancy in protein
identification, and the FDR was kept less than 1%. When the same peptide(s) were
assigned to multiple proteins, the multiple proteins were clustered into a “protein
group”. Furthermore, the protein with the highest sequence coverage in each “protein
group” was extracted for bioinformatics analysis.
Ⅱ. Characterization
a
b
c
Figure S1. SEM images of b-MIPs (a), silica mold (b), and h-MIPs particles (c).
a
b
Figure S2. IR spectrum of silica (a) and h-MIP microspheres (b). After the removal of
silica mold (a), strong characteristic Si-O bands at 1103.3cm-1 disappeared in the
h-MIP microspheres (b).
Table S1. Yields of polymer prepared by different pore sizes of the silica particles
Pore size
particles
of
the
Yield
silica
MIP
NIP
300Å
———
100mg/g
500Å
50mg/g
100mg/g
1000Å
70mg/g
300mg/g