## 4 Material and Methods
### Isotope Coded Protein Labeling (ICPL)  
Before ICPL-labeling, acetone precipitation of solubilized and pooled proteins from microdissected (LCM) tissue was performed [10; 11]. To one volume of each sample 5 volumes of 100% acetone cooled to \(-20\)\({}^{\circ}\)C were added and incubated overnight at \(-20\)\({}^{\circ}\)C. Precipitated proteins were pelleted by centrifugation 30 min at 20,000\(\times\)\(g\) at 4 \({}^{\circ}\)C. Pellets were washed twice with cold 80% (\(v/v\)) acetone by centrifugation for 5 min at 20,000\(\times\)\(g\) at 4 \({}^{\circ}\)C. The protein concentration was determined by a Bradford assay (BioRad Laboratories GmbH, Munich, Germany) before and after acetone precipitation, the last of which was used to adjust sample concentration at 5 \(\upmu\)g/\(\upmu\)L in ICPL-lysis buffer (40 \(\upmu\)L) containing 6 M guanidine-HCL and 0.1 M HEPES pH 8.5. For ICPL-labeling, cysteines were reduced by adding of 1 \(\upmu\)L of 2 M TCEP in 0.1 M HEPES pH 8.5 into each of the 4 samples and overfilled with argon. After vortexing for 30 s and 1 min ultrasonic treatment, the samples were incubated for 30 min at 60 \({}^{\circ}\)C. For alkylation of cysteines 1 \(\upmu\)L of 0.4 M IAA (Iodoacetamide) in 0.1 M HEPES pH8.5 was added into each of the 4 samples and incubated for 30 min at 25 \({}^{\circ}\)C. Alkylation was quenched by adding 0.5 M  
Figure 3: Laser capture micodissection of tumor biopsies. (**A**) The tumor tissue is circled by a green line; (**B**) the laser dissects the tumor tissue which will be picked up and separated for proteomic analysis. Scale bar = 300 \(\upmu\)m, numbers are starting points for the laser. N-acetyl cysteine solution in 0.1 M HEPES pH 8.5 followed by incubation for 15 min at 25 \({}^{\circ}\)C. For the differential labeling, four ICPL-reagents, ICPL\({}_{0}\), ICPL\({}_{4}\), ICPL\({}_{6}\) and ICPL\({}_{10}\), were used as aquadruplex approach. Proteins were then labeled with one of the 4 ICPL reagents (6 \(\upmu\)L of 0.15 M ICPL\({}_{0}\), ICPL\({}_{4}\), ICPL\({}_{6}\) or ICPL\({}_{10}\)). The reaction was allowed to proceed for 2 h at 25 \({}^{\circ}\)C under a nitrogen atmosphere to minimize methionine oxidation. Labeling was quenched by addition of hydroxylamine (1.5 M, pH 8.3), and all four samples were combined and stored by \(-\)20 \({}^{\circ}\)C. From each of the two separately prepared technical replicates, the sample amount was divided for the OFFGEL-fractionation (95%; 150 \(\upmu\)g) and for non-fractionation (5%; 10 \(\upmu\)g). For OFFGEL-fractionation, ICPL-labeled and combined proteins (150 \(\upmu\)g) were fractionated by isoelectric point (pJ) using an Agilent 3100 OFFGEL fractionator (Agilent Technologies, Oberhaching, Germany) according to the manufacturer's instructions The samples from the first technical replicate (24 OFFGEL-fractions and non-fractionated sample) were analyzed after in-solution digestion with the enzymes Glu-C und Trypsin [17] by the Core Facility (Max-Planck Institute for Biochemistry, Martinsried, Germany) with the LTQ-hybrid mass spectrometer (Thermo Fischer Scientific, Bremen, Germany). Nano-LC-MS/MS was performed on an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Schwerte) coupled to a nanoHPLC Agilent 1200 system (Agilent Technologies Deutschland GmbH, Boblingen, Germany). Briefly, peptides were preconcentrated on a reversed-phase (RP) trapping column (15 cm-fused silica column with 75 \(\upmu\)m ID filled with ReproSil-Pur C18-AQ 3 \(\upmu\)m resin (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) in 0.1% TFA (Trifluoroaectic) using a gradient from 2% to 40% in 0.5% acetic acid in ACN (Acetonitrile) at a flow rate of 250 nL/mL in 100 min. MS (Mass Spectrometry) survey scans were acquired within the Orbitrap from 300 to 1800 \(m/z\) at a resolution of 60.000. The ten most intense signals were subjected to collision induced dissociation (CID) in the ion trap.