| Metadata |
| datasetIdentifier | PASS00632 |
| datasetType | SRM |
| submitter | Alexander Schäfer <alexander.schaefer@helmholtz-muenchen.de> |
| submitter_organization | Helmholtz Center Munich |
| lab_head_full_name | Prof. Dr. Marius Ueffing |
| lab_head_email | marius.ueffing@uni-tuebingen.de |
| lab_head_organization | Helmholtz Center Munich AND Institute for Ophthalmic Research at the University of Tübingen |
| lab_head_country | Germany |
| datasetTag | Mouse_Liver_T2D |
| datasetTitle | Mouse Liver Proteins implicated in Metabolism and Type 2 Diabetes. |
| publicReleaseDate | 2014-12-09 00:00:00 |
| finalizedDate | 2014-12-09 06:45:30 |
| summary | While the performance of LC-SRM applied to samples from various bodily fluids, particularly plasma, and microorganisms has been extensively investigated,there is only little experience with its application to animal tissue samples.
Here, we show that a conventional one-dimensional LC-SRM workflow applied to mouse liver tissue suffers from a shortcoming in terms of sensitivity for lower abundance proteins. This problem could be solved through the extension of the standard workflow by an additional dimension of separation at the peptide level prior to online LC-SRM. For this purpose, we used off-gel electrophoresis (OGE) which is also shown to outperform strong cation exchange (SCX) in terms of resolution, gain of signal intensity, and predictability of separation. The extension of the SRM workflow by a high resolving peptide separation technique is an ideal combination as it allows the addition of stable isotope standards directly after trytic digestion and will increase the dynamic range of protein abundances amenable by SRM in animal tissue. |
| contributors | Alexander Schäfer, Christine von Törne, Silke Becker, Hakan Sarioglu, Melanie Kahle, Susanne Neschen, Stefanie M. Hauck, Marius Ueffing |
| publication | PMID:22994301
Anal Chem. 2012 Oct 16;84(20):8853-62. doi: 10.1021/ac3023026. Epub 2012 Oct 4.
Two-dimensional peptide separation improving sensitivity of selected reaction monitoring-based quantitative proteomics in mouse liver tissue: comparing off-gel electrophoresis and strong cation exchange chromatography.
Schäfer A1, von Toerne C, Becker S, Sarioglu H, Neschen S, Kahle M, Hauck SM, Ueffing M. |
| growth | Mouse Liver Samples: C3HeB/FeJ female mice were bred
in house and housed in isolated ventilated cages (IVC-Racks,
BioZone) supplied with filtered air, in a 12/12 h light/dark cycle
(lights on from 6 am until 6 pm). Mice had free access to food
and water. |
| treatment | At 8 weeks of age, mice were killed and organs were
perfused with 10 mL of ice-cold 0.9% NaCl via heart. Liver tissue
samples were snap-frozen in liquid nitrogen and stored at −80
°C. All procedures for animal handling and experiments were
performed in accordance with protocols approved by the
Regierung von Oberbayern (District Government of Upper
Bavaria). In vivo experiments were conducted in the German
Mouse Clinic phenotyping platform (GMC) at the Helmholtz
Center Munich. |
| extraction | Frozen liver samples were ground to powder using mortar and pestle under
liquid nitrogen and stored at −80 °C until use. Powdered liver
samples were mixed with extraction buffer (RIPA, 50 mM, Tris
150 mM NaCl, 0.1% (w/v) sodium dodecyl sulfate (SDS), 0.5%
(w/v) deoxycholate, 1% (v/v) NP40, 1× protease inhibitors) in
CKM homogenization tubes (Precellys lysing kit, Bertin
Technologies) on ice. Proteins were extracted by bead-based
mechanical homogenization in a Precellys 24 Homogenizer
(Bertin Technologies) prechilled to 0 °C by 25 s cycles of
grinding at 5500 rpm. Homogenates were cleared by
centrifugation at 16 000g for 30 min at 4 °C, and protein
concentration was determined by BCA assay (Pierce) according
to the manufacturer’s instructions. |
| separation | Off-Gel Electrophoresis (OGE) of Peptides: OGE was
performed on an Agilent 3100 OFFGEL Fractionator with an
optimized protocol using consumables from Agilent: IPG strips
(pH 3−10, linear, 7 cm, GE Healthcare) were swollen in 30 mL
of H2O overnight at RT. The next day, strips were dehydrated in
10 mL of acetonitrile for 20 min at RT. Dehydrated strips were
inserted into off-gel trays, fixed with frames, which had been
clipped to 7 wells, and rehydrated using 100 μL of 0.2%
ampholytes pH 3−10 in H2O per well for 20 min at RT. Peptides
were diluted in 525 μL of 0.2% ampholytes pH 3−10 in H2O and
applied to the strip at 75 μL/well. Isoelectric focusing was
conducted with the settings: 2000 V, 50 μA, 200 mW, and 12 °C
until 15 kVh was reached. Fractions were collected, and wells
were rinsed with 100 μL of 80% methanol, 0.5%TFA for 5 min to
improve peptide retrieval. Corresponding fractions were pooled.
Highly acidic and basic peptides were extracted from electrode
papers using 100 μL of 50% acetonitrile (ACN), 0.1% TFA and
combined with the most acidic and most basic fractions,
respectively. Organic solvents were removed by 45 min vacuum
centrifugation at 35 °C, and fractions were concentrated using
PepClean (Pierce) according to the manufacturer's instructions
to a final volume of 20 μL. Fractions five and six, counted from
the anode, were pooled. Average pH values were measured on a
strip run without peptides in parallel using a micro pH electrode
(Mettler Toledo).
Strong Cation Exchange (SCX) Separation of Peptides: Off-line SCX fractionation of peptides was conducted on an
Ettan micro-HPLC (GE Healthcare) equipped with a C18 trapcolumn
(1 × 5mmPepMap100 C18, 5 μm, 100 Å; LC Packings)
and an SCX analytical column (0.3 × 150 mm PolysulfethylA
SCX, 5 μm, 300 Å, PolyLC) in backflush configuration. Peptides
were loaded onto the trap column in 0.1% TFA and desalted at
30 μL/min for 10 min. Transfer of peptides from C18 to SCX
was done by a short organic bump using 80% acetonitrile, 0.1%
formic acid in water at 3 μL/min for 3 min. Subsequently,
peptides were separated using 5% acetonitrile, 0.1% formic acid
in water (A) and 5% acetonitrile, 0.1% formic acid, 500mMNaCl
in water (B) at a flow rate of 5 μL/min, according to the following
gradient schedule: 0−5 min, 2% B; 5−25 min, 2−30% B; 25−30
min, 30−65% B; 30−31 min, 65−95% B, hold 95% for 6 min and
re-equilibrate at 2% B for 15 min. Peptide elution was monitored
at 214 nm, and 5 min fractions were collected. |
| digestion | In Solution Digestion (Trypsin). For mouse liver extracts,
acid labile detergent rapigest (Waters) was added to a final
concentration of 2% (w/v) and protein samples were denatured
and reduced using DTT at a final concentration of 3.5 mM at 60
°C for 10 min. Cysteines were alkylated using IAA at a final
concentration of 11.25 mM for 30 min at RT in the dark.
Subsequently, samples were diluted 10-fold with 50mMTris pH
8.5 and digested with trypsin at 1:20 enzyme/protein for 18 h at
37 °C. Afterward, rapigest was hydrolyzed by lowering the pH
below 2 for 30 min on ice. As indicated in the results, in some
instances, peptides were further purified using PepClean C18
spin column (Pierce) according to manufacturer’s instructions
but loading no more than 10 μg of peptide on one column. |
| acquisition | LC-SRM/MS analysis was performed on a Tempo nano MDLC system (AB Sciex) coupled online to a QTrap 4000 (AB Sciex) mass spectrometer by a nano spray III ion source. Peptides were loaded onto a nano trap column (0.3 × 5 mm, PepMap100 C18, 5 μm, 100 Å; LC Packings) in 0.1% TFA at a flow rate of 20 μL/min for 5 min and separated on a C18 analytical column (0.075 × 150 mm, PepMap100 C18, 3 μm, 100 Å, LC Packings) by a gradients of 90min using 2%acetonitrile in 0.1%formic acid in water (A) and 0.1% formic acid in 98% acetonitrile (B) at a flow rate of 250 nL/min. The gradient settings were: 5−65 min, 5−40% B; 65−70 min, 40−90% B; 70−72 min, 90% B; 72−79 min, 90−5% B, reequilibration for 10 min. Electrospray ionization was maintained with curtain gas set to 14 psi, a spray voltage of 2.6kV, ion source gas set to 30 psi, and an interface heater temperature of 170 °C. Peptides were detected in SRM mode with Q1 and Q3 set to unit resolution and dwell times ranging from 20 to 50 ms.Validated SRM assays were combined into one multiplex method in which peptide measurements were conducted at unit resolution in Q1 and Q3 using scheduled SRM with a retention time window and a target scan time adjusted gradient length according to observed peptide separation characteristics. Retention time window and target scan time were 2 min/5s. |
| informatics | Raw data acquired using Analyst 1.5 was imported into Multiquant 2.0 (AB
Sciex) for quantitative analysis. Peak areas were determined by
integration of SRM traces with the MQ4 algorithm using the
following settings: Gaussian smooth width, 3 points; min peak
width, 3 points; noise percentage, 85%; baseline subtraction
window, 2 min; peak splitting, 2 points. Signal-to-noise ratios
were determined using the SignalFinder algorithm with the
settings: confidence threshold, 50%; no global baseline, allow
nonlinear baseline.
Visual comparison of SRM traces was done with Skyline 1.2
using Savitzky−Golay smoothing of all traces. |
| instruments | Q-Trap 4000/Eksigent MDLC |
| species | Mouse |
| massModifications | C+57.021464
K+8.014199
R+10.008269 |
