Supporting information
Proteinase K improves quantitative acylation studies
Benjamin Fränzel1, Frank Fischer2, Clemens Steegborn2, Dirk Andreas Wolters1*
1 Dept.
of Analytical Chemistry, Biomolecular Mass Spectrometry,
Institute of Chemistry and Biochemistry, Ruhr-Universität Bochum, 44801 Bochum
Dept. Biochemistry, NW I, University of Bayreuth, 95447 Bayreuth
*Corresponding author:
Dirk A. Wolters
Department of Analytical Chemistry
Biomolecular Mass Spectrometry
Ruhr University Bochum
NC 4/72
Universitaetsstr. 150
44801 Bochum, Germany
Tel
+ 49 (0) 234/32-25463
Fax
+ 49 (0) 234/32-14742
Email
Dirk.Wolters@ruhr-uni-bochum.de
METHODS
Chemicals
Ammonium bicarbonate (AmBic), ammonium carbonate, ammonium hydroxide, ammonium
sulphate, ammonium acetate, formic acid (FA), trifluoric acid (TFA), hydrochloric acid,
methanol, and HPLC grade acetonitrile (ACN) were purchased from Mallinckrodt Baker
(Deventer, The Netherlands). Sequencing grade trypsin was procured from Promega
(Madison, WI). Modified sequencing grade chymotrypsin was purchased from Princeton
Separations INC. (Adelphia, NJ). Proteinase K was acquired from Roche Diagnostics
(Mannheim, Germany). Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from
PAN-Biotech (Aidenbach, Germany). Histones, compounds for phosphate buffered saline
(PBS), and TWEEN20 were from Sigma-Aldrich (St. Gallen, Switzerland) and anti acetyl-lysine
antibodies were obtained from Immunechem (Burnaby, Canada). Stable isotope labeled
amino acids were purchased from Silantes (Munich, Germany).
Growth and Cell Lysis of HEK293
HEK293 were cultivated in 75 cm² cultivation flasks at 37°C in DMEM medium for at least 5
doubling times. Cells were harvested by centrifugation at 5,000 x g for 10 min, washed once
with PBS buffer and re-suspended in PBS lysis buffer with 0.1 mL protease inhibitors (Sigma
Aldrich, Germany). Sonification was used to disrupt the cells. Cell debris was removed at 500
x g for 10 min and discarded. Subsequently, proteins were precipitated in 80% acetone. At
least three technical replicates were scrutinized for each experiment. For SILAC experiments,
13C-lysine
and 13C/15N-arginine were utilized. Thereby, wild type was compared with a Sirtuin
isoform 5 overexpressor cell line, kindly provided by Marcia Haigis (Harvard Medical School).
This cell line was cultivated on heavy SILAC medium, whereas wild type was cultivated on
light SILAC medium, respectively.
Digestion Conditions
For optimization of the digestion condition, test analyses were conducted with 1 mg purified
histones (H1.1, H2A, H3.1, H3.3 and H4.1) from calf (Sigma) for each test, which were outlined
in triplicates. LC-MS/MS experiments used linear increasing ACN-gradients over 90 minutes.
Three technical replicates were analysed from each fraction. To 100 μg protein amount
sequencing grade trypsin and chymotrypsin (1:50 w/w) were supplied in 50 mM ammonium
bicarbonate buffer. The preparation was incubated at 37°C overnight, centrifuged at 15,000
x g and finally diluted in water and 0.1% formic acid.
Proteinase K digestion was performed in 100 mM ammonium carbonate buffer for 1 hour at
room temperature 1:100 w/w). Afterwards, digestion was stopped with 0.1% TFA and
solvent was evaporated.
For the direct analysis 100 μg whole cell proteins were precipitated with acetone and
incubated in 50 mM ammonium bicarbonate buffer plus 20% methanol using sequencing
grade trypsin (1:50 w/w) overnight at 37°C. After centrifugation at 15,000 x g peptides were
finally diluted in water and 0.1% formic acid.
Enrichment of Acetylated Peptides
For enrichment of acetylated peptides, agarose coupled anti acetyl-lysine antibodies were
used according to the supplier’s manual. Briefly, 20 µL agarose antibodies were washed with
PBST buffer (PBS containing 0.02% TWEEN20). Binding of acetylated peptides from 1 mg
protein mixture was achieved in 2 hours at room temperature. Subsequently, antibodies
were washed and bound peptides were finally eluted using 0.5 N hydrochloric acid.
For SILAC analysis, 1 mg of HEK cell lysate from wt and Sirt-5 overexpressor cells were each
added to 100 mM ammonium carbonate buffer and 10 µg proteinase for 1 hour at room
temperature. Afterwards, digestion was stopped with 0.1% TFA and solvent was evaporated.
LC-MS/MS and MudPIT Analysis
A quaternary Accela U-HPLC pump was connected to a Thermo LTQ Orbitrap Velos from
Thermo Fisher Scientific Inc. (Waltham, MA) ion trap mass spectrometer. The digested
samples were loaded onto a biphasic micro-capillary pre-column (diameter = 100 μm), which
was equipped with an in-house manufactured frit, 5 cm strong cation exchange SCX material
(Phenomenex, Germany) and with 5 cm of Luna C18 RP material (Phenomenex, Germany).
The main chromatographic column was packed with 45 cm of Luna C18 RP material
(Phenomenex, Germany) and held in a thermo-stated metal block at 45°C. The two columns
were connected with a PEEK union. The joined columns were connected to a PEEK microcross to split the flow of the HPLC pump to an effective flow rate of 150 – 250 nL/ min and
supply a spray voltage of 1.8 kV. The LTQ Orbitrap Velos was operated using instrument
method files in the Sequence Setup window of Xcalibur. The heated desolvation capillary
was set to 200°C. For a 10 step MudPIT experiment, each step was represented by one
instrument method file with identical settings except for the gradient program for the HPLC
pump. The instrument method files of the LTQ Orbitrap were set to detect a full MS scan
between 400 and 2000 m/z for the precursor ion (resolution = 60,000) followed by full
MS/MS scans of the top 20 ions from the preceding MS scan to detect fragment ions by the
ion trap.
Data Analysis
To interpret MS/MS data we used the SEQUEST algorithm implemented in Proteome
Discoverer 1.2. Data were searched against a Uniprot human database (2007) containing
17,482 protein entries. All accepted results had a false discovery rate (FDR) of 0.8 and lower
determined by decoy database search. Singly, doubly, and triply charged peptides had a
cross-correlation score (XCorr) above 1.8, 2.5 or 3.5. Furthermore, mass accuracy was set to
10 ppm and the oxidation of methionine was admitted as variable peptide modification.
Additionally, the RSp value was set to 1. A protein was considered as present in the mixture
when two or more unique peptides were identified by the previous criteria.
For acetylation studies, acetylated lysine was set as variable modification. Unspecific
database searches (no enzyme specification) for large datasets were performed using the
SEQUEST algorithm implemented in the multi-thread compatible search engine ProLuCID.
Protein quantification was performed using the ProRata 1.0 algorithm. We reported earlier
how we performed the quantification using the ProRata algorithm (Fränzel B, Fischer F,
Trötschel C, Poetsch A, Wolters D (2009) The two-phase partitioning system--a powerful
technique to purify integral membrane proteins of Corynebacterium glutamicum for
quantitative shotgun analysis. Proteomics 9(8):2263–2272). Briefly, the minimum log2 ratio
was set to -7 the maximum log2 ratio to 7, respectively. Each protein was quantified by at
least two different peptides and the left peak shift and the right peak shift were set to zero
scans. We considered a protein as significantly regulated at a log2 value of at least 1 and -1
respectively. From four biological replicates at least two had to show regulation by the
previous criteria to ascertain significance.
a)
b)
c)
Figure S1: The elution profile of peptides shown in Figure 4 of the main text are displayed. Light and
heavy peptides are eluting simultaneously.
Sequence
Log2R
Log2SNR
Validity
a)Y.SLKNQIGDKEKLGGK#L
2.17067
2.85496
true
b)L.KNQIGDKEKLGGK#L.S
0
0
false
c)L.KNQIGDKEKLGGK#LS.S
2.12134
2.13646
true