Supporting Information Methods
An automated in-gel digestion / iTRAQ-labeling workflow for robust quantification of
gel-separated proteins
Carla Schmidt, Dörte Hesse, Monika Raabe, Henning Urlaub, Olaf Jahn
Gel electrophoresis
Proteins were separated on precast NuPAGE 4-12 % Bis-Tris gels (Invitrogen) using a MES
buffer system and visualized with standard colloidal Coomassie staining. Dilutions of LMW
marker (GE Healthcare) were used as source for the standard proteins BSA and ovalbumin.
GRAVY index of BSA and ovalbumin was computed with the ProtParam tool
(http://web.expasy.org/protparam) by using their processed sequences.
LC-MS/MS analysis of tri-snRNP samples
Labeled peptides were separated by nano-flow reversed-phase liquid chromatography (HP
1100 series, Agilent; mobile phase A, 0.1% (v/v) formic acid (FA); mobile phase B, 95%
(v/v) ACN/0.1% (v/v) FA) coupled to a LTQ-Orbitrap XL hybrid mass spectrometer
(Thermo Scientific). The peptides were loaded onto a trap column packed in-house (2 cm,
360 μm o.d., 150 μm i.d.; ReproSil-Pur, C18, AQ 5 µm, Dr. Maisch HPLC GmbH) and
separated with a flow rate of 300 nL/min on an analytical C18 capillary column (15 cm, 360
μm o.d., 75 μm i.d.; ReproSil-Pur, C18, AQ 3 µm, Dr. Maisch HPLC GmbH), with a gradient
of 0-38% (v/v) mobile phase B over 30 min.
MS/MS analysis in CID mode. Typical mass spectrometric conditions were: spray voltage of
1.8 kV; capillary temperature of 150 °C; normalized collision energy of 37.5 % at an
activation of q = 0.25 and an activation time of 35 ms. The LTQ-Orbitrap XL was operated in
data-dependent mode. Survey full scan MS spectra were acquired in the orbitrap (m/z
350−1600) with a resolution of 30,000 at m/z 400 and an automatic gain control (AGC) target
at 106. The five most intense ions were selected for CID MS/MS fragmentation in the linear
ion trap at an AGC target of 100,000. Detection in the linear ion trap of previously selected
ions was dynamically excluded for 60 s. Singly charged ions as well as ions with
unrecognized charge state were also excluded. Internal calibration of the orbitrap was
performed using the lock mass option (lock mass: m/z 445.120025; Olsen JV et al., Mol Cell
Proteomics 2005, 4:2010-21.)
MS/MS analysis in HCD mode. Typical mass spectrometric conditions were: spray voltage of
1.8 kV; capillary temperature of 150 °C; normalized collision energy of 40.0 % at an
activation time of 40 ms. The LTQ-Orbitrap was operated in data-dependent mode. Survey
full scan MS spectra were acquired in the orbitrap (m/z 300−1400) with a resolution of
30,000 at m/z 400 and an automatic gain control (AGC) target at 106. The five most intense
ions were selected for HCD MS/MS fragmentation in the HCD cell and detection in the
orbitrap with a resolution of 7500 at m/z 400. Detection of previously selected ions was
dynamically excluded for 30 s. Singly charged ions as well as ions with unrecognized charge
state were also excluded. Internal calibration of the orbitrap was performed using the lock
mass option (lock mass: m/z 445.120025; Olsen JV et al., Mol Cell Proteomics 2005, 4:201021.)
LC-MS/MS of nuclear extract samples
Labeled peptides were separated by nano-flow reversed-phase liquid chromatography (HP
1100 series, Agilent; mobile phase A, 0.1% (v/v) formic acid (FA); mobile phase B, 95%
(v/v) ACN/0.1% (v/v) FA) coupled to a LTQ-Orbitrap Velos mass spectrometer (Thermo
Scientific). The peptides were loaded onto a trap column packed in-house (2 cm, 360 μm o.d.,
150 μm i.d.; ReproSil-Pur, C18, AQ 5 µm, Dr. Maisch HPLC GmbH) and separated with a
flow rate of 300 nL/min on an analytical C18 column packed in-house into a picofritcolumn
(New Objective) (12 cm, 360 μm o.d., 75 μm i.d.; ReproSil-Pur, C18, AQ 3 µm, Dr. Maisch
HPLC GmbH), with a gradient of 3-90 % (v/v) mobile phase B over 38 min.
MS/MS analysis in HCD mode. Typical mass spectrometric conditions were: spray voltage of
1.8 kV; capillary temperature of 270 °C; normalized collision energy of 45.0 % at an
activation time of 0.1 ms. The LTQ-Orbitrap was operated in data-dependent mode. Survey
full scan MS spectra were acquired in the orbitrap (m/z 350−1600) with a resolution of
30,000 at m/z 400 and an automatic gain control (AGC) target at 106. The 10 most intense
ions were selected for HCD MS/MS fragmentation in the HCD cell and detection in the
orbitrap with a resolution of 7500 at m/z 400. Detection of previously selected ions was
dynamically excluded for 60 s. Singly charged ions as well as ions with unrecognized charge
state were also excluded. Internal calibration of the orbitrap was performed using the lock
mass option (lock mass: m/z 445.120025; Olsen JV et al., Mol Cell Proteomics 2005, 4:201021.)
Database search and data analysis
Labeled tri-snRNP samples. Raw data were searched against NCBI non-redundant (2011-0506, 11961441 sequences) or NCBI Reference Sequence human (2009-11-11, 37871
sequences) databases using the Mascot v2.2.06 or v2.3.02 search engine (Matrix Science).
For NCBInr database search, the taxonomy filter Homo Sapiens (human) was used. The mass
accuracy filter was 5 ppm for precursor ions and 0.5 Da for MS/MS fragment ions. Peptides
were defined to be tryptic with maximal two missed cleavage sites. Carbamidomethylation of
cysteine residues and oxidation of methionine residues were allowed as variable
modifications. For testing the labeling efficiency, iTRAQ modification of lysine and tyrosine
residues as well as peptide N-termini were allowed. For quantification experiments, iTRAQ
modification of tyrosine residues were allowed as variable modification and iTRAQ
modification of lysine residues and peptide N-termini were considered as fixed modifications.
Labeled nuclear extract samples. Raw data were searched against the NCBI non-redundant
(2011-09-13, 15270974 sequences) database restricted to the taxonomy Homo Sapiens
(human) using the Mascot v2.3.02 search engine (Matrix Science). The mass accuracy filter
was 10 ppm for precursor ions and 0.6 Da for MS/MS fragment ions. Peptides were defined
to be tryptic with maximal two missed cleavage sites. Carbamidomethylation of cysteine
residues and oxidation of methionine residues were allowed as variable modifications. For
testing the labeling efficiency, iTRAQ modification of lysine and tyrosine residues as well as
peptide N-termini were allowed. For quantification experiments, iTRAQ modification of
tyrosine residues were allowed as variable modification and iTRAQ modification of lysine
residues and peptide N-termini were considered as fixed modifications. Only peptides with
MS/MS ions score >30 were considered.
iTRAQ quantification
Peptide ratios were derived from the peak areas of iTRAQ-115, -116, and -117 reporter ions,
each divided by that of iTRAQ-114 reporter ion (internal standard). The respective reporter
ion ratios 115/114, 116/114 and 117/114 were obtained from Mascot. Reporter ion ratios
<0.09 and >11 were considered as indicative of incomplete labeling and manually deleted
from the dataset. Protein ratios were calculated by dividing the sum of the peptide ratios by
the number of peptide ratios: Protein ratio = (ΣPeptide ratios)/(#Peptide ratios). Protein ratios were
normalized by setting the smallest ratio to one and calculating the other ratios accordingly.