Super-SILAC allows Classification of Diffuse Large B-cell ...

amino acids and supplemented with 20% dialyzed fetal bovine serum. Cells were cultured for at least 6
passages until they were fully labeled as assessed by quantitative mass spectrometry. Less than 1% of
tryptic peptides contained unlabeled arginine or lysine in the 9 labeled cell lines (Ramos, Mutu, Bl-41,
U2932, OciLy3, BJAB, L1236, L428, DB) and less than 0.3% of identified peptides showed evidence of Arg
to Pro conversion. Equal amounts of the heavy lysates were mixed to generate the super-SILAC mix.
Protein Digestion and Fractionation – The super-SILAC mix (100 µg) was combined with an equal amount
of the unlabeled cells and further processed by the filter-aided sample preparation (FASP) method (21).
In short, the sample was loaded on Microcon filters with a 30 kDa cut-off (Millipore, Billerica, MA, USA),
which allows the replacement of SDS with a urea containing buffer. The proteins were then alkylated
with iodoacetamide followed by overnight trypsin digestion at 37°C in 50 mM ammonium bicarbonate.
Peptides were collected from the filter after centrifugation and elution with water (2x).
Using strong anion exchange chromatography, 40 �g of the peptide mixture from each replicate was
fractionated (22). In summary, the strong anion exchange (SAX) was performed in tip-columns prepared
from 200 μl micropipet tips stacked with six layers of a 3 M Empore Anion Exchange disk (1214–5012
Varian, Palo Alto, CA). We used Britton & Robinson universal buffer (BRUB) composed of 20 mM acetic
acid, 20 mM phosphoric acid, and 20 mM boric acid and titrated with NaOH to the desired pH for
column equilibration and fraction elution. After loading the peptides at pH 11 and collecting it, five
additional fractions were collected consecutively with buffers of pH 8, 6, 5, 4, and 3. The eluted fractions
were desalted on reversed phase C18 Empore disc StageTips (23). Peptide elution was performed twice
with 20 μl buffer B containing 80% acetonitrile (ACN) in 0.5% acetic acid. Organic solvents were removed
by a SpeedVac concentrator to prepare the samples for analysis by LC-MS/MS.
Liquid chromatography and MS for fractionation experiments – Eluted peptides were separated on an
in-house-made 15 cm column with 75 µm inner diameter packed with ReproSil-Pur C18-AQ 3 μm resin
(Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) using an Easy nanoflow high-performance liquid
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