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Supplementary Data Supplemental Material Materials and Methods Immunohistochemistry Primary antibodies used for validation studies include: mouse anti-desmoglein-3 (Cat. # 32-6300, Invitrogen, CA, USA; 1:25), rabbit anti-cytokeratin 4 (Cat. # ab11215, Abcam, Cambridge, MA, USA; 1:100), mouse anti-cytokeratin 16 (Cat. # ab8741, Abcam; 1:25), rabbit anti-desmoplakin antibody (Cat. # ab14418, Abcam; 1:200), mouse anti-vimentin (Cat. # M7020, Dako, Carpinteria, CA, USA; 1:100). Secondary antibodies conjugated with biotin (Vector, Burlingame, CA, USA) were used, diluted to 1:400. Tissues slides containing archival FFPE sections, or tissue micro arrays (TMA) consisting of 508 HNSCC and controls, were dewaxed in SafeClear II (Fisher Scientific, Pittsburgh, PA, USA) hydrated through graded alcohols, immersed in 3% hydrogen peroxide in PBS for 30 min to quench the endogenous peroxidase, and processed for antigen retrieval and immunostaining with the appropriate primary antibodies and biotinylated secondary antibodies as described (1), followed by the avidin-biotin complex method (Vector Stain Elite, ABC kit; Vector). Slides were washed and developed in 3,3'- diaminobenzidine (Sigma FASTDAB tablet; Sigma Chemical) under microscopic control, and counterstained with Mayer's hematoxylin. For each stained TMA the number of positive cells in each core was visually evaluated and the results expressed as a percentage of stained cells/ total number of cells. According to their immunoreactivity the tissues array cores were divided according to tumor differentiation, where the percentage of stained cells in the three tumor classes were scored as more than 5% and less than 25% of the cells stained, 26 to 50%, 51 to 75% or, 75 to 100%. Patel et al., 1 Laser Capture Microdissection (LCM) FFPE oral cancer tissue sections were deparaffinized in SafeClear II and hydrated through graded alcohols to 70% ethanol. Slides were briefly washed in water and stained in Mayer’s hematoxylin (10-15 sec) followed by dehydration through graded ethanol (70-100%) and SafeClear II. For LCM, stained uncovered slides were air dried, and cells of interest captured using a PIXCELL IIe (Molecular Devices Corporation, Sunnyvale, CA, USA) as described (2). Approximately 20,000 cells were capture from a single or consecutive tissue sections using up to 4 CapSure LCM Caps (Molecular Devices Corporation), which were transferred to a 0.5 ml sterile Eppendorf tube for protein extraction (see below). Protein Extraction Protein extraction was carried out using the Liquid Tissue MS Protein Prep kit according to manufacturer’s protocol (Expression Pathology Inc., Gaithersburg, MD, USA). Briefly, the films from the underside of the caps for each sample were removed, transferred to low binding reaction tubes, and incubated with 20 µl of Liquid Tissue extraction buffer at 950C for 1.5 h, with intermittent mixing followed by a quickspin, to consolidate the material. Samples were centrifuged at 10,000 rpm for 1 min, cooled on ice, followed by digestion with 1 µl of trypsin (15-20 U/µl) for 1 h at 370C with intermittent mix and spin. The samples were centrifuged one final time at 10,000 rpm for 1 min prior to reduction with 2 µl of 100 mM DTT (10 mM final concentration) at 950C for 5 min, followed by centrifugation and storage at –80 0C until ready for analysis. Patel et al., 2 Tandem Mass Spectrometry and Bioinformatic Analysis FFPE extracted samples were acidified with 0.1% trifluoroacetic acid (TFA) and desalted with C-18 ZipTip microcolumns (Millipore, Billerica, MA, USA), lyophilized to dryness, resuspended in 0.1% TFA, and quantified by the BCA protein assay (Pierce, Rockford, IL, USA). Nanoflow reversed-phase liquid chromatography (RPLC) was performed using an Agilent 1100 capillary LC system (Agilent Technologies, Palo Alto, CA, USA) coupled online to a linear ion trap (LIT) mass spectrometer (LTQ, Thermo Electron, San Jose, CA, USA). Separations were performed using 75 µm i.d. x 360 o.d. x 10 cm long fused silica capillary columns (Polymicro Technologies, Phoenix, AZ, USA) that were slurry packed in house with 3 µm, 300 Å pore size C-18 silica-bonded stationary phase (Vydac, Hysperia, CA, USA). After injecting 2 µL of sample (approximately 200 ng protein), the column was washed for 30 min with 98% mobile phase A (0.1% formic acid in water) at a flow rate of 0.5 µL/min. Peptides were eluted using a linear gradient of 2% mobile phase B (0.1% formic acid in acetonitrile) to 40% solvent B in 110 minutes, then to 98% B in an additional 30 min, all at a constant flow rate of 0.25 µL/min. The LIT-MS was operated in a data dependent MS/MS mode in which each full MS scan is followed by five MS/MS scans where the five most abundant peptide molecular ions are selected for collision-induced dissociation (CID), using a normalized collision energy of 35%. Dynamic exclusion was utilized to minimize redundant selection of peptides previously selected for CID. The heated capillary temperature and electrospray voltage were set at 160 ºC and 1.6 kV, respectively. Data were collected over a broad mass to charge (m/z) precursor ion selection scan range of 400-2000, followed by four segmented precursor selection scan ranges (e.g. gas Patel et al., 3 phase fractionation in the m/z dimension, GPFm/z) using the following overlapping m/z intervals: 400-605, 595-805, 795-1205, and 1195-2000. Tandem mass spectra were searched against the UniProt human protein database (06/2005 release) from the European Bioinformatics Institute (http://www.ebi.ac.uk/integr8) using SEQUEST (Thermo Electron). For a fully tryptic peptide to be considered legitimately identified, it had to achieve stringent charge state and proteolytic cleavage-dependent cross 1+ 2+ 3+ correlation (Xcorr) scores of 1.9 for [M+H] , 2.2 for [M+2H] and 3.5 for [M+3H] , and a minimum delta correlation ∆Cn) of 0.08. Results were further filtered using software developed in-house to determine unique peptides and proteins. Based on criteria followed in previous work, the error in protein identification was <1.5% (3). To facilitate biological interpretation of the protein lists derived from this experiment, data was subjected to EASE analysis (Expression Analysis Systematic Explorer) to identify Gene Ontology (GO) categories and terms that might indicate over-representation in the tissues analyzed. References 1. Amornphimoltham P, Patel V, Sodhi A, et al. Mammalian target of rapamycin, a molecular target in squamous cell carcinomas of the head and neck. Cancer Res 2005;65:9953-61. 2. Baker H, Patel V, Molinolo AA, et al. Proteome-wide analysis of head and neck squamous cell carcinomas using laser-capture microdissection and tandem mass spectrometry. Oral Oncol 2005;41:183-99. 3. Hood BL, Darfler MM, Guiel TG, et al. Proteomic analysis of formalin-fixed prostate cancer tissue. Mol Cell Proteomics 2005;4:1741-53. Patel et al., 4 Supplemental Table 1: Proteins identified in only in normal squamous epithelium Peptides Accession Protein (number) Q12756 Kinesin-like protein KIF1A (Axonal transporter of synaptic vesicles) 6 P35813 Protein phosphatase 2C alpha isoform (EC 3.1.3.16) (PP2C-alpha) (IA) (Protein phosphatase 1A) 3 Q9Y561 Low-density lipoprotein receptor-related protein 12 precursor (Suppressor of tumorigenicity protein 7) 3 Q6ZN03 Hypothetical protein FLJ16545 3 O94916 Nuclear factor of activated T cells 5 (T cell transcription factor NFAT5) (NF-AT5) (Tonicity-responsive 2 P23458 Tyrosine-protein kinase JAK1 (EC 2.7.1.112) (Janus kinase 1) (JAK-1) 2 P13611 Versican core protein precursor (Large fibroblast proteoglycan) (Chondroitin sulfate proteoglycan core 2 P16455 Methylated-DNA--protein-cysteine methyltransferase (EC 2.1.1.63) (6-O-methylguanine-DNA methyltra 2 Q9H0M3 Hypothetical protein DKFZp434O0710 (Hypothetical protein KIAA1549) 2 Q9UGU5 High-mobility group protein 2-like 1 (HMGBCG protein) 2 Q6ZWK7 Hypothetical protein FLJ16045 2 Q7Z7J3 Hypothetical protein 2 Q6UXX9 QFRL9384 2 Q96BY6 Dedicator of cytokinesis protein 10 (Protein zizimin 3) 2 O43693 Zinc-finger protein (Fragment) 1 P68106 FK506-binding protein 1B (EC 5.2.1.8) (Peptidyl-prolyl cis-trans isomerase 1B) (PPIase 1B) (Rotamase 1B) 1 P68402 Platelet-activating factor acetylhydrolase IB beta subunit (EC 3.1.1.47) (PAF acetylhydrolase 30 kDa sub 1 P78318 Immunoglobulin-binding protein 1 (CD79a-binding protein 1) (B cell signal transduction molecule alpha 4) 1 O15537 Retinoschisin precursor (X-linked juvenile retinoschisis protein) 1 P57082 T-box transcription factor TBX4 (T-box protein 4) 1 O43300 Leucine rich repeat neuronal protein 2 precursor (Leucine-rich repeat transmembrane neuronal 2 protein 1 P62906 60S ribosomal protein L10a (CSA-19) 1 P62690 HERV-K_22q11.23 provirus ancestral Gag polyprotein (Gag polyprotein) [Contains: Matrix protein; Capsid 1 P51813 Cytoplasmic tyrosine-protein kinase BMX (EC 2.7.1.112) (Bone marrow kinase BMX) (Epithelial and endoth 1 O60235 Airway trypsin-like protease precursor (EC 3.4.21.-) 1 P52961 GPI-linked NAD(P)(+)--arginine ADP-ribosyltransferase 1 precursor (EC 2.4.2.31) (Mono(ADP-ribosyl)tra 1 Q13445 Putative T1/ST2 receptor binding protein (Interleukin 1 receptor-like 1 ligand,) 1 P05937 Calbindin (Vitamin D-dependent calcium-binding protein, avian-type) (Calbindin D28) (D-28K) 1 Q14674 Separin (EC 3.4.22.49) (Separase) (Caspase-like protein ESPL1) (Extra spindle poles-like 1 protein) 1 Q13945 3',5'-cyclic AMP phosphodiesterase (Fragment) 1 Q13591 Semaphorin 5A precursor (Semaphorin F) (Sema F) 1 Q14202 Zinc finger protein 261 (DXS6673E protein) 1 P82650
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