Supplementary informations
Proteome analysis of testis from infertile protein C inhibitor deficient mice
reveals novel changes
in serpin processing and prostaglandin metabolism
Authors:
Hanjiang Yang1, Felix Christof Wahlmüller1, Pavel Uhrin1, Roland Baumgartner2, Goran
Mitulovic3, Bettina Sarg4, Margarethe Geiger1 and Maria Zellner2
Affiliations:
1 Center
of Physiology and Pharmacology, Department of Vascular Biology and Thrombosis Research,
Medical University of Vienna, Austria
2 Center
of Physiology and Pharmacology, Institute of Physiology, Medical University of Vienna,
Austria
3Clinical
Department of Laboratory Medicine Proteomics Core Facility, Medical University of Vienna
4Division
of Clinical Biochemistry, Biocenter, Innsbruck Medical University, Innrain 80-82, 6020
Innsbruck, Austria
*
Corresponding author:
Maria Zellner, Institute of Physiology, Center of Physiology and Pharmacology, Medical
University of Vienna, Schwarzspanierstrasse 17, 1090 Vienna, Austria
E-mail: maria.zellner@meduniwien.ac.at;
Tel.: +43-1-40160-31420; Fax: +43-1-40160 9623
Supplementary informations 1 2D-DIGE analysis of mouse testes
For alkaline proteins, Immobiline IPG-DryStrips (24 cm, pH6-9, GE Healthcare) were
rehydrated in 450 µl modified rehydration solution (7 M urea, 2 M thiourea, 150 mM DTT, 2%
ampholytes pH6-9). Isoelectric focusing (IEF) using an IPGphor unit (GE Healthcare) was
completed after cup-loading of 15 µg Cy2-, Cy3-, and, C5-labeled protein samples each and
reaching 30 kVh. Resolution of acidic proteins (24 cm, pH4-7, GE Healthcare) was
accomplished passively with in-gel rehydration of 45 μg (three samples, 15 μg each) CyDyelabelled protein extracts in a similar rehydration buffer substituted with 70 mM DTT and 1%
ampholytes, pH4-7 (Serva, Heidelberg, Germany). IEF was performed as described above.
Proteins were separated in the second dimension using 11.5% SDS-PAGE (35 V for 1 h, 50
V for 1.5 h, and finally 110 V for 16.5 h at 10 °C) and images with a resolution of 100 µm
were acquired on a Typhoon 9410 imager (GE-Healthcare, Freiburg, Germany). The
DeCyder software module ‘Differential In-gel Analysis’ (version 6.00.28; GE Healthcare) was
applied to detect spots on the images with a target spot number of 5000. All sample gels
were matched with the master gel in the DeCyder module ‘Biological Variation Analysis’
(version 6.01.02).. The standardised abundance (SA) of a protein spot in each sample was
calculated relative to the same spot of the internal standard (pool of all samples from 2DIGE
analysis experiment) to reduce inter-gel variation.
Supplementary informations 2
2.1 Protein identification by mass spectrometry from Goran Mitulovic
Tryptic peptides were re-dissolved in 40 µl of 0.1% aqueous TFA, and 20 µl of the peptide
solution was diluted with 30µl 0.1% TFA. Out of this solution 40 µl were injected onto the
nano separation column. The nano HPLC separation system was an UltiMate nano HPLC
(LC Packings, Amsterdam, The Netherlands) consisting of Famos autosampler, Switchos
column switching unit, and the UltiMate nano pump and the UV detector. The Acclaim C18
trap column (300 µm ID x 5mm) was operated at the ambient temperature, and the Acclaim
C18 nano separation column (75 µm ID x 250 mm) separation column was mounted in the
column oven and operated at 45°C. Samples were loaded onto the trap column using 0.1%
TFA at 30 µl/min, and the separation column was operated in gradient mode at 300 nl/min.
User Defined Injection Program (UDP) was used for sample injection and additional injector
and trap column wash. Every sample injection was followed by two runs with injections of
2,2,2-trifluoroethanol (TFE) to remove possible sample remains in injector and on the trap
column, and prevent carry-over in the separation system. The separation gradient was
generated as described in Table 1S by mixing following solvents:
A: 95% aqueous 0.1% formic acid (FA); 5% acetonitrile,
B: 70% aqueous 0.1% FA; 15% acetonitrile; 15% methanol, and
C: 0.8% FA in 60% acetonitrile; 30% methanol; 10% (TFE).
Time (min)
0
8
45
65
70
72
85
Table 1S LC Separation gardient
%B
3
3
100
0
0
3
3
%C
0
0
0
100
100
0
0
Mass spectrometric analysis was performed using the LTQVelos IonTrap mass spectrometer
(ThermoFisher, Bremen, Germany) with the “Top 20” method: the twenty most intensive ions
from the MS scan were selected for MS/MS. Single charged ions were excluded from
fragmentation and detected ions were excluded for further fragmentation for three minutes
after initial MS/MS fragmentation have been performed. In addition, prior to the MS, UV
peptide detection at 214 nm was also performed.
Data analysis was performed using Mascot (Matrix Science, London, UK) and searching the
most recent version of the SwissProt database. All search results were refined and
researched using Scaffold 3.6.2 (www.proteomesoftware.com).
2.2 Protein identification by mass spectrometry analysis from Bettina Sarg
Protein digests were analyzed using an UltiMate 3000 nano-HPLC system (Dionex,
Germering, Germany) coupled to an LTQ Orbitrap XL mass spectrometer (ThermoScientific,
Bremen, Germany) equipped with a nanospray ionization source [1]. A homemade fritless
fused silica microcapillary column (75 µm i.d. x 280 µm o.d.) packed with 10 cm of 3 µm
reverse-phase C18 material (Reprosil) was used. The gradient (solvent A: 0.1% formic acid;
solvent B: 0.1% formic acid in 85% acetonitrile) started at 4% B. The concentration of solvent
B was increased linearly from 4% to 50% during 50 min and from 50% to 100% during 10
min. A flowrate of 250 nL/min was applied.
MS instrument settings were as follows: Mass spectra were acquired in positive ion mode
applying a data-dependent automatic switch between survey scan and MS/MS acquisition.
Survey MS scans (from m/z 300 – 2000) were acquired in the Orbitrap with a resolution of
R = 60,000; the target value was 200,000; maximum ionization time was set to 20 ms. Up to
four of the most intense ions per scan were fragmented in the linear trap (LTQ) using
collision induced dissociation (CID) at a target value of 10,000; the maximum ionization time
was set to 100 ms. Dynamic exclusion settings: repeat count 1; repeat duration 50 sec;
exclusion duration was 45 sec. Charge state screening was enabled; unassigned and singly
charged ions were excluded from MS/MS.
MS2 spectra were searched against a NCBInr mouse database via SEQUEST, Proteome
Discoverer software (Version 1.1, ThermoScientific). Database search criteria were set as
follows: enzyme, trypsin; maximum miss cleavage sites, 2; precursor tolerance, 10 ppm;
fragment
mass
tolerance,
0.8
Da.
The
variable
modifications
considered
were
carbamidomethylation (+57.021) at Cys and oxidation (+15.995) at Met. All data were filtered
to satisfy a false discovery rate (FDR) of 1% or better.
References
[1] Sarg, B., Faserl, K., Kremser, L., Halfinger, B., Sebastiano, R., Lindner, H. H., Molecular
& cellular proteomics : MCP 2013, 12, 2640-2656.