Download Interface Solution Isoelectric Focusing with in situ MALDI

Interface Solution Isoelectric Focusing with in situ
MALDI-TOF Mass Spectrometry
Shujun Wang,1, 2 Suming Chen,3 Jianing Wang,3 Peng Xu,2 Yuanming Luo,2 Zongxiu Nie,3,*
Wenbin Du2, 1*
Supporting Information
Chemicals and Materials: All water used was deionized water from a Hitech Master-S UVF
Water Purification System (Shanghai, China). Ribonuclease A from Bovine Pancreas (RNase
A, MW: 13.7 kDa, pI=7.8) was obtained from Sangon Biotech. (Shanghai, China). Green
Fluorescent Protein (GFP, MW: ~29 kDa, pI=4.7~4.9) was obtained from Sino Biological Inc.
(Beijing, China).
Bovine Serum Albumin (BSA, MW: ~66 kDa, pI=5.6), Fluorescein
isothiocyanate (FITC), Sinapic acid (SA), and 1H, 1H, 2H, 2H-Perfluoro-1-octanol (PFO) were
obtained from Sigma Aldrich. Pharmalyte (pH 3-10) was purchased from GE Healthcare
(Sweden). Methylcellulose and Tetramethylethylenediamine (TEMED) were purchased from
Yuanju Biotech. (Shanghai, China). α-cyano-4-hydroxycinnamic acid (CHCA) was purchased
from
TCI
Company
(Shanghai,
China).
Tridecafluoro-1,1,2,2-tetrahydrooctyl-1-
trichlorosilane was obtained from Fluorochem Ltd. (Derbyshire, UK). FC-40 oil was obtained
from 3M (St. Paul, MN).
Soda-lime glass (0.7 mm thick) with chromium and AZ1500 photoresist coating were
purchased from Telic Company (Valencia, CA). Amorphous diamond coated drill bits were
obtained from Harvey Tool (Rowley, MA). Teflon tubing (O.D. 150 μm, I. D. 100 μm) was
purchased from Zeus (Orangeburg, SC). Conductive tape was obtained from Kaivo
Optoelectronic Technology Co. (Zhuhai, China).
Instruments and Computer Programs: Pipettes were obtained from Eppendorf (Hamburg,
Germany). Stereoscope was purchased from Shunyu Optical Technology (Ningbo, China). A
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custom made DC power supply was used for IEF. A transilluminator with an excitation
wavelength of 470 nm and an emission wavelength of 510 nm was purchased from Zeesan
Biotechnology (Xiamen, China). All fluorescence images were taken on a Nikon Eclipse-Ti
inverted fluorescence microscope (Tokyo, Japan). Images were analyzed with ImageJ software.
MALDI-TOF MS analysis of samples was carried out on a Bruker ultrafleXtreme instrument
(Bruker Daltonics, USA) equipped with a 355 nm Nd-YAG laser, ion acceleration voltage of
25 kv and pulse extraction delay time of 283 ns. Each spectrum obtained was the summation
of 500 laser shots to maximize the signal to noise ratio. MS results were analyzed with Bruker
Daltonics FlexAnalysis 4.0 Software and MALDI Imaging MS was conducted and analyzed
with Bruker Daltonic fleximaging 3.0 software.
FITC-labeling procedure for proteins: FITC labeling was carried out according to the
product instructions. Briefly, proteins were dissolved in 40 mmol/L carbonate buffer to
concentration of 10 mg/mL. FITC solution (1.5 mg/mL) was prepared in 40 mmol/L carbonate
buffer. Then FITC solution was slowly added into the protein solution in the dark with stirring.
After that, the mixture was incubated in the dark with stirring at 4 ºC for 12 hours. Finally, the
reaction product was purified with a dialysis bag (MWCO: 4000 Da) in 1X PBS for 24 hours
to remove the excess FITC and other substances with low molecular weight.
Depositing of matrix: Matrix for MALDI-TOF MS analysis were prepared by dissolving
matrix in 50% acetonitrile, 49.9% D. I. water, and 0.1% trifluoroacetic acid. MALDI matrix
solutions for protein samples and the CSF sample were 10 mg/mL sinapic acid (SA) and 5
mg/mL α-cyano-4-hydroxycinnamic acid (CHCA) respectively.
Commercial-available Matrix Sprayers can be used for applying matrix on the glass plate
automatically. In this work, 10-nL matrix solution was manually deposited into each microwell
for co-crystallization with the sample. A 50 µL Hamilton glass syringe was placed on a Harvard
Apparatus syringe pump (Holliston, MA). The syringe was connected with a piece of Teflon
tubing (150 μm O. D., 100 μm I. D., 10 cm long) and filled with matrix solution. The tip of the
2
tubing was placed in the microwells under stereoscope, and matrix solution was injected into
each microwell with a preset target volume of 10 nL at a flow rate of 0.3 μL/min.
3
Figure S1. The slipping and freezing procedure prior to MS detection. a-e) Cross-section
schematics of the device operation after sIEF prior to MS; f-g) Photographs of the microwell
array containing blue dye solution slipped to the etched open area with and without the freezing
step.
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Figure S2. Steps for generate identical IEF segments on the two glass plates. The device (a)
was slipped horizontally to overlap the microwell array on the top plate and bottom plate (b),
and slipped again to divide the mixed microwells and generate duplicates (c).
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Figure S3. Photograph of the glass plates with conductive copper tapes on the MALDI target
plate. White cross on the plate are markers for alignment.
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Figure S4. Mass spectra of CSF sample indicating that there are no metabolites with molecular
weight larger than 600. The CSF sample were prepared same as sIEF experiments (added with
carrier ampholyte, TEMED and glycerol, and cocrystallized with CHCA). The data are overlay
of 14 Mass spectra each was the summation of 500 laser shots to enhance to maximize the
signal to noise ratio.
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Table S1. Peaks observed in mass spectra with mass-to-charge ratio (m/z) from 100 to 600.
No.
(1) control solution of
carrier ampholyte
(m/z)
(2) directly deposited CSF
sample with carrier
ampholyte (m/z)
1.
2.
3.
100.904
102.828
114.932
100.845
102.787
114.893
4.
116.884
116.880
5.
(3) CSF sample
separated by sIEF on chip
(m/z)
100.9200
pI
--
114.6197
--
116.4879
--
123.4551)
--
130.8108
--
6.
130.967
130.920
7.
144.719
144.967
8.
146712
146.973
147.0020
--
9.
150.172
150.573
150.1157
--
10.
159.053
159.135
158.8340
--
11.
172.774
173.059
172.5342
--
12.
176.174
--
13.
176.2706
--
186.85712)
5.09
14.
190.838
190.5935
--
15.
213.064
213.0120
--
16.
234.80783)
--
17.
236.13004)
--
18.
264.07645)
--
19.
275.28566)
--
20.
295.21327)
--
21.
296.50908)
--
22.
303.30889)
--
23.
319.499910)
--
24.
336.313811)
25.
337.777012)
--
26.
347.523013)
--
27.
372.692
172.84
372.4325
28.
374.013014)
--
29.
378.659015)
4.9
30.
380.528116)
--
31.
382.104017)
--
32.
403.569318)
--
33.
406.879
83.7
406.6830
34.
408.197019)
--
35.
420.657020)
--
36.
442.801721)
--
37.
475.183922)
--
38.
(4) peak heights ratio
of column (3) over
column (2)a)
481.029
65.87
480.7886
39.
503.829823)
4.6
40.
515.661824)
--
41.
556.762425)
--
42.
559.876026)
--
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1)-26)
a)
26 new Peaks were discovered after the sIEF-MALDI SlipChip;
Those peaks observed in the control solution, and peaks observed exclusively on sIEF
SlipChip were not calculated.
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Table S2. Prediction of possible metabolites in the CSF microdialysate sample based on the
peaks obtained by MALDI imaging (in positive ion mode).
m/z of peaks
Possible metabolites
Average molecular weight HMDB ID a)
a
116.699
L-Proline
115.1300
HMDB00162
147.404
Acetylcholine
146.2074
HMDB00895
L-Lysine
146.1876
HMDB00182
148.023
L-Glutamic acid
147.13
HMDB00148
174.950
L-Arginine
174.201
HMDB00517
176.394
N-Acetylaspartic acid (NAA)
175.14
HMDB00812
186.865
Phosphoserine
185.0725
HMDB00272
189.141
Homo-L-arginine
188.2275
HMDB00670
N6,N6,N6-Trimethyl-L-lysine
188.2673
HMDB01325
337.759
Kyotorphin
337.3742
HMDB05768
556.762
Enkephalin L
555.6227
HMDB01045
The Human Metabolome Database (HMDB), http://www.hmdb.ca/
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