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Proteomics Methodology
Fast protein liquid chromatography
CP samples were collected by manual dissection and sonicated in 0.065% trifluoroacetic acid
(TFA). Total protein contained in 400 μl of this buffer was filtered through a 0.22-µm membrane
(Millipore) and injected into an AKTA Purifier FPLC system (Amersham Biosciences) for separation
through a reverse phase FPLC Resource RPC column (3 ml, Amersham Biosciences). Flow rate was set
at 2 ml/min (Solution A was .065% TFA and solution B was 0.05% TFA+84% acetonitrile). Protein was
eluted by a 0-80% gradient of solution B. All run parameters were controlled by Unicorn V4.0 software.
The fraction collector was set to collect protein elution peaks into individual collection tubes based on
in-line peak sensing. A total of 40 tubes were collected and individually processed for enzymatic
digestion and mass spectrometry based analysis.
Two dimensional chromatofocusing/non-porous reverse phase HPLC protein separation
Two dimensional liquid chromatography (2D LC) was performed on a Beckman-Coulter
Proteomelab PF2D System. The CP was at 1mg/ml in 0.5M TEB/0.1% SDS buffer. An acetone
precipitation was performed by reducing the volume to 50µl, adding 450µl cold acetone containing
1mM HCL, and incubating at -20ºC for 3 hours. After microfuging, the pellet was washed 2 x with cold
acetone and dissolved in 1ml of chromatofocusing buffer A. The first dimension (chromatofocusing)
was on a 2.1 x 250 mm HPCF-1D column (Eprogen, Inc.) using a pH monitor, UV 280nm trace, and
Buffers A (25 mM Bis-Tris, 6 M urea, and 0.2% n-octyl β-D-glucopyranoside, pH 8.5) and B
(polybuffer 74 diluted 1:10 with water, 6 M Urea, and 0.2% n-octyl β-D-glucopyranoside, pH 4.0). The
entire 1mg of sample was analyzed using a 115 minute 0-100% pH gradient at 200 µl/min with fractions
collected at 0.3-pH intervals. The second dimension (nonporous reverse-phase HPLC separation) was
performed at 50°C on a 4.6 x 33 mm HPCF2D column (Eprogen, Inc.) equilibrated with 0.1% TFA
(Buffer A). The first dimension fractions were separately injected (20 fractions) and intact proteins
eluted using a 30 minute 0-100% Solvent B (100% Acetonitrile, 0.08% TFA) linear gradient. Proteins
were detected at 214 nm with 450 fractions collected. Fractions were pooled based on the UV trace into
216 fractions for LC-MS/MS analysis.
In gel digestion
Proteins isolated in 1 dimensional polyacrylamide gels were subjected to in situ enzymatic
digestion. The gel plugs were washed with 250l 50% acetonitrile/50% water for 5 minutes followed by
250l of 50mM ammonium bicarbonate/50% acetonitrile/50% water for 30 minutes and 250µl 10mM
ammonium bicarbonate/50% acetonitrile/50% water for 30 minutes. After washing, the gel plugs were
dried in a Speedvac, rehydrated with 0.1g modified trypsin (Promega)15mm3 of gel in 15 l 10mM
ammonium bicarbonate and digested at 37 C for 16 hours.
In solution digestion
Proteins isolated from the 2D LC, C18 reverse phase, and FPLC fractionation were Speedvac
dried prior to dissolving in 10 l 8M urea, 0.4M ammonium bicarbonate. Samples were reduced by the
addition of 5 l 45mM dithiothreitol (DTT) and incubating at 37C for 20 minutes. Alkylation was
done using 5 l of 100 mM iodoacetamide (IAN) and incubating at ambient temperature for 20 minutes.
The urea concentration was decreased to 2M with the addition of water and Promega sequencing grade
trypsin added at a 1:15 weight to weight ratio. Digestion proceeded for 16 hours at 37C. For a double
digestion, the endopeptidase Lys-C (Wako) was added at a 1:15 weight/weight ratio, digested for 5-16
hours, and then followed by the trypsin addition as above.
Strong Cation Exchange Peptide Separation
After digestion, the digest was acidified (2µl 1M phosphoric acid) and strong cation exchange
chromatography was performed using a 2.1x 200mm PolySULFOETHYL A™ column (PolyLC Inc.) on
an Applied Biosystems Vision Workstation. A linear 118 minute gradient (Buffer A: 10mM potassium
phosphate, 25% acetonitrile pH 3.0 and Buffer B: 10mM potassium phosphate, 25% acetonitrile pH 3.0,
plus 1M potassium chloride) was run. Each cation-exchange fraction (20 fractions) was dried and resuspended in 5ul 70% formic acid, diluted to 15µl with 0.1% TFA prior to injecting 5µl on Thermo
Scientific LTQ Orbitrap XL mass spectrometer.
LC-MS/MS
Sample digestions (except the C18 reverse phase intact protein samples) were subjected to LCMS/MS on a Thermo Scientific LTQ Orbitrap XL equipped with a Waters nanoAcquity UPLC system,
with a Waters Symmetry® C18 180µm x 20mm trap column and a 1.7 µm, 75 µm x 250 mm
nanoAcquity™ UPLC™ column (35ºC) for peptide separation. Trapping was done at 15µl/min, 99%
Buffer A (100% water, 0.1% formic acid) for 1 min. Peptide separation was performed at 300 nl/min
with Buffer A: 100% water , 0.1% formic acid and Buffer B: 100% CH3CN, 0.075% formic acid. A
linear gradient (51 minutes) was run with 5% buffer B at initial conditions, 50% B at 50 minutes, and
85% B at 51 minutes. MS was acquired in the Orbitrap using 1 microscan, and a maximum inject time
of 900 followed by 4 to 6 data dependant MS/MS acquisitions in the ion trap. Neutral loss scans (MS3)
were also obtained for 98.0, 49.0, and 32.7 amu for the titanium dioxide enriched fractions. The data
was searched using Mascot Distiller and the Mascot search algorithm.
LC-MS/MS on the C18 reverse phase intact protein digests was performed on an Applied
Biosystems QSTAR XL or QSTAR Elite interfaced with a LC Packings Ultimate workstation or a
Waters nanoACQUITY respectively. For the QSTAR XL, a 5mm C18 LC Packings PepMap trap
column was used with RP-HPLC at a flow rate of 400 nl/min on a 100 um x 15 cm Waters Atlantis C18
column C-18 column. Initial HPLC conditions were 95% buffer A and 5% buffer B with the following
linear gradient: 3 min, 5% B; 43 min 37% B; 75 min 75% B; and 85 min 95% B. Buffer A consisted of
98% water, 2% acetonitrile, 0.1% acetic acid, and 0.003% TFA. Buffer B contained 80% acetonitrile,
20% water, 0.09% acetic acid, and 0.003% TFA. For the QSTAR Elite, the Waters nanoACQUITY was
operated under the same conditions as for the LTQ Orbitrap.
Database searching
MS/MS spectra were searched in-house using the Mascot algorithm (Hirosawa et al, 1993,
version 2.2.0) on a MASCOT Server 2.2 (Linux, Cluster) after using the Mascot Distiller program to
generate Mascot compatible files. All LC-MS/MS data were searched against the IPI Rat database
(EMBL-EBI) and a protein was considered identified when (1) Mascot listed it as significant, (2) more
than 2 peptides match the same protein, and (3) > 95% probability. Typical parameters used for
searching were partial methionine oxidation, carboxamidomethylated cysteine, and phosphorylation.
For the LTQ Orbitrap runs, a peptide tolerance of +20ppm, a MS/MS fragment tolerance of +0.6 Da,
and peptide charges of +2 or +3 were used. The QSTAR XL and Elite runs used a peptide tolerance of
0.4 Da and 0.6 Da for MS/MS.
Normal and decoy database searches were run.
Bioinformatics analysis of CP proteome
The CP proteome was compared with proteomes from kidney tubules, kidney glomeruli,
cerebellum and lateral ventricle glia. Protein data sets were obtained from the well annotated Human
Protein Atlas (http://www.proteinatlas.org/). Only proteins that had an annotated expression reliability
score of “high” were used. Comparisons were made for statistical enrichment of particular gene
ontology (GO) molecular function, biological process terms, transcription factors and protein interaction
partners. MetaCore (GeneGO, Inc.) was used for the analysis.