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Fast and Simple Protocols for Mass Spectrometry-Based Proteomics of Small Fresh FrozenUterine Tissue Sections
Dapic, I.; Uwugiaren, N.; Jansen, P.J.; Corthals, G.L.
Published in:Analytical Chemistry
DOI:10.1021/acs.analchem.7b01937
Link to publication
Citation for published version (APA):Dapic, I., Uwugiaren, N., Jansen, P. J., & Corthals, G. L. (2017). Fast and Simple Protocols for MassSpectrometry-Based Proteomics of Small Fresh Frozen Uterine Tissue Sections. Analytical Chemistry, 89(20),10769-10775. https://doi.org/10.1021/acs.analchem.7b01937
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Download date: 23 Mar 2021
Supporting Information
S-1
Fast and simple protocols for mass spectrometry-based proteomics
of small fresh frozen uterine tissue sections
Irena Dapic1, Naomi Uwugiaren1, Petra J. Jansen1 and Garry L. Corthals1
1University of Amsterdam, Van 't Hoff Institute for Molecular Sciences, Analytical-
Chemistry Group, Science Park 904, 1098 XH Amsterdam, The Netherlands
Corresponding author: Prof. Garry L. Corthals, University of Amsterdam, Van 't Hoff
Institute for Molecular Sciences, Analytical-Chemistry Group, Science Park 904, 1098
XH Amsterdam, The Netherlands; E-mail: G.L.Corthals@uva.nl, Phone:
+31202525406
Supporting Information
Supporting Information
S-2
Table of content
Figure S-1: Instrumental parameters.......................................................................S-3
Figure S-2: Qualitative reproducibility......................................................................S-4
Figure S-3: Quantitative reproducibility;;;;;;;...........................................S-5
Figure S-4: Identified proteins and peptides;.........................................................S-8
Figure S-5: Sequence coverage analysis................................................................S-9
Figure S-6: NSAF analysis.....................................................................................S-10
Supporting Information
S-3
Figure S-1. Influence of the instrumental parameters on number of identified proteins
and peptides. Results are displayed for optimization of instrumental parameters for
both TOF-MS and TOF-MS/MS accumulation time. Data were recorded in intensity
dependent acquisition (IDA) mode with the following parameters: TOF-MS (m/z: 400-
1250; threshold 100 cps; top ions: 30 with charge from +2 to +4; exclusion time: 30
s); TOF-MS/MS (m/z: 200-1800). Other parameters regarding instrumental settings
were as described in Experimental section.
%
0 20 40 60 80 100
Proteins
Peptides
MS: 50 ms; MS/MS: 100 ms
MS: 250 ms; MS/MS: 100 ms
MS: 250 ms; MS/MS: 250 ms
MS: 500 ms; MS/MS: 100 ms
Supporting Information
S-4
a)
b)
c)
d)
Figure S-2. Venn diagrams showing qualitative reproducibility of the proteins
between 3 biological replicates for 10, 16 and 20 µm tissue (a-c); shared proteins
for 10 µm and 16 µm thick tissue identified from 3 replicates for Protocol 1 and 2
(d).
Supporting Information
S-5
a)
Protocol 1
Protocol 2
Protocol 3
Protocol 4
Protocol 5
Supporting Information
S-6
b)
Protocol 1
Protocol 2
Protocol 3
Protocol 4
Protocol 5
Supporting Information
S-7
c)
Figure S-3. Quantitative reproducibility of the proteins between 3 biological replicates
of FF human uterus tissue. Results are shown as correlation of the NSAF values of
the identified proteins for (a) 10, (b) 16 and (c) 20 µm thick FF human uterus tissue.
Protocol 2
Protocol 3
Protocol 4
Protocol 5
Supporting Information
S-8
a)
b)
Figure S-4. Number of identified proteins (a) and peptides (b) using different
extraction and digestion conditions. Proteins and peptides were identified at 1% FDR
in 10, 16 and 20 µm thick tissue after protein extraction and digestion using Protocols
1 to 5. Results are shown as mean±SD.
Pro
toco
l 1
Pro
toco
l 2
Pro
toco
l 3
Pro
toco
l 4
Pro
toco
l 5
Pro
toco
l 1
Pro
toco
l 2
Pro
toco
l 3
Pro
toco
l 4
Pro
toco
l 5
Pro
toco
l 2
Pro
toco
l 3
Pro
toco
l 4
Pro
toco
l 5
Num
ber
of id
entified p
eptides
Proto
col 1
Proto
col 2
Proto
col 3
Proto
col 4
Proto
col 5
Proto
col 1
Proto
col 2
Proto
col 3
Proto
col 4
Proto
col 5
Proto
col 2
Proto
col 3
Proto
col 4
Proto
col 5
Supporting Information
S-9
a)
b)
c)
Figure S-5. Results from the study evaluated for the sequence coverage of the
proteins extracted from 3 biological replicates distributed according to their Mw: a)
10 µm, b) 16 µm and c) 20 µm thick FF human uterus tissue. Results are shown
as mean±SD.
Mw /kDa
0-10
10-2
0
20-3
0
30-4
0
40-5
0
50-6
0
60-7
0
70-8
0
80-9
0
90-1
00
>100
0
10
20
30
40
Protocol 1
Protocol 4
Protocol 5
Protocol 2
Protocol 3
Mw /kDa
0-10
10-2
0
20-3
0
30-4
0
40-5
0
50-6
0
60-7
0
70-8
0
80-9
0
90-1
00
>100
0
10
20
30
40
Protocol 1
Protocol 4
Protocol 5
Protocol 2
Protocol 3
Mw /kDa
0-10
10-2
0
20-3
0
30-4
0
40-5
0
50-6
0
60-7
0
70-8
0
80-9
0
90-1
00
>100
0
10
20
30
40
Protocol 4
Protocol 5
Protocol 2
Protocol 3
Supporting Information
S-10
a)
b)
c)
Figure S-6. NSAF values of the proteins extracted from 3 biological replicates
distributed according to their Mw: a) 10 µm, b) 16 µm and c) 20 µm thick FF
human uterus tissue. Results are shown as mean±SD.
0-10
10-2
0
20-3
0
30-4
0
40-5
0
50-6
0
60-7
0
70-8
0
80-9
0
90-1
00
>100
0-10
10-2
0
20-3
0
30-4
0
40-5
0
50-6
0
60-7
0
70-8
0
80-9
0
90-1
00
>100
0-10
10-2
0
20-3
0
30-4
0
40-5
0
50-6
0
60-7
0
70-8
0
80-9
0
90-1
00
>100
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