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Metadata
datasetIdentifierPASS00254
datasetTypeMSMS
submitterHari Kosanam <hamallik@gmail.com>
submitter_organization
lab_head_full_name
lab_head_email
lab_head_organization
lab_head_country
datasetTagPancreaticTumors
datasetTitlePROTEOMIC ANALYSIS OF PANCREATIC ADENOCARCINOMA TISSUES
publicReleaseDate2013-07-01 00:00:00
finalizedDate2013-06-10 17:13:50
summarywe performed Orbitrap® mass spectrometry proteomic analysis of four pancreatic ductal adenocarcinoma tissues and their adjacent benign tissues and identified a total of 2,190 non-redundant proteins.For all four patients included in this study, the PDAC tumors were surgically resected from the head region of the pancreas. Their age/sex/clinical diagnosis-TNM classification was: 59/F/pT3 pN1 cM0 G2 R0; 57/F/pT3 pN1 cM0 G3 R1; 75/F/pT3 pN1 cM0 G3 R1; 56/M/pT3 pN0 cM0 G2 R0. Four benign tissues were collected adjacent to their PDAC counterparts. Tissues were fixed in Tissue-TEK® shortly after their surgical removal.
contributorsHari Kosanam and Eleftherios P. Diamandis
publicationHari Kosanam, Ioannis Prassas, Caitlin C. Chrystoja, Ireena Soleas, Alison Chan, Apostolos Dimitromanolakis, Ivan M. Blasutig, Felix Rückert, Robert Gruetzmann, Christian Pilarsky4, Masato Maekawa5, Randall Brand, and Eleftherios P. Diamandis
LAMC2: A PROMISING NEW PANCREATIC CANCER BIOMARKER
IDENTIFIED BY PROTEOMIC ANALYSIS OF PANCREATIC ADENOCARCINOMA

Submitted to Molecular Cellular Proteomics; MCP/2012/023507
growth
treatment
extractionThe frozen specimens were thawed to room temperature and washed several times with phosphate buffered saline (PBS-pH 7.4) to remove the TEK material. The clean tissues were transferred to sterilized 1.5 mL Eppendorf® tubes. Tissues were pulverized using a sterile glass rod and ~ 0.5 mL of T-PER® (Thermo Scientific, Mississauga, Canada) was added (T-PER is a detergent-based tissue lysis reagent). The mixture was sonicated for about 5 min on ice using MISONIX immersion tip sonicator (Q SONICA LLC, CT, USA). Tissue protein extracts were centrifuged to remove debris and subjected to overnight dialysis (1 kDa molecular weight cutoff membrane) to remove salts and surfactants. The dialyzed tissue extracts were measured for total protein content using the BCA assay (Thermo Scientific, Mississauga, Canada). Equal amounts (~500 µg) of protein from eight samples (four PDAC and four controls) were subjected to tryptic digestion.
separationTryptic-peptides were acidified in buffer A (0.26 M FA in 5% acetonitrile) and loaded onto a PolySULFOETHYL aspartamide strong cation exchange (SCX) column (4.6mm x 50mm, 200A and 5µm) (The Nest Group, Inc., MA, USA) and fractionation was performed using an Agilent 1100 HPLC system. A 60 min linear gradient method was operated with buffer A→B (B: 0.26 M FA in 5% acetonitrile and 1 M ammonium formate) at a flow rate of 250µL/min. Fractions were collected in 250µL aliquots which were later pooled into 12 fractions.
digestion
acquisitionThe peptides from SCX fractions were desalted using the Omix C18 tips (Varian Inc., Palo Alto, CA, USA). Samples were diluted with Buffer A (0.1 % FA in water) and injected onto a C18 trap column (150 µm; packed in-house) using the EASY-nLC system (Proxeon Biosystems, Odense, Denmark). Peptides were eluted from the trap column with an increasing concentration of Buffer B (0.1% FA in acetonitrile) onto a resolving 5 cm long PicoTip Emitter (75 µm inner diameter, 8 µm tip, New Objective) packed in-house with 3 µm Pursuit C¬18 (Varian Inc.). Peptides were resolved using gradient reverse phase (RP) liquid chromatography at a flow rate of 400 nL/min for 90 min. The chromatography system was connected online to an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) via a nano-ESI source (Proxeon). The capillary temperature was 160oC and spray voltage was 2 kV. The mass spectra were acquired in data-dependent mode. The first scan event was a full MS scan from 450-1450 m/z and the next six scans were MS/MS scans on the six most intense parent ions observed in the first scan. These scan events were alternated between each other for 90 min MS acquisition time. Collision dissociation energy for MS/MS was set at 35%. Dynamic exclusion, monoisotopic precursor selection and charge state screening were enabled
informaticsThe MS spectra were searched against the non-redundant IPI human database (version 3.71 containing 86745 forward and 86745 reverse protein sequences) using two search engines, separately: Mascot, version 2.1.03 (Matrix Science) and the Global Proteome Machine manager, version 2006.06.01 (GPM X! Tandem; Beavis Informatics Ltd., Canada). The following parameters were used: (I) enzyme: trypsin; (II) one missed cleavage allowed; (III) fixed modification: carbamidomethylation of cysteines; (IV) variable modifications: oxidation of methionines; (V) MS1 tolerance, 7 ppm; and (VI) MS2 tolerance, 0.4 Da. The resulting Mascot DAT and X! Tandem XML files were merged using Scaffold® (version 2.06, Proteome Software Inc., Portland, Oregon) with ‘MudPIT’ (multidimensional protein identification technology) option checked. Scaffold data was filtered using the X! Tandem Log E (min 3.0) and Mascot ion-score filters [ion score 15, 30(+2) and 40 (+3)] in order to obtain a protein false-positive rate (FPR) of ≤ 1%. FPR = 2 X (number of proteins identified by searching the reverse sequences)/ (the total number of identified proteins). Scaffold® protXML reports were exported and uploaded into Protein Center (Proxeon Biosystems, Odense, Denmark) to create Venn diagrams.
instrumentsThermo Scientific LTQ Orbitrap
speciesHuman
massModificationsstatic: C+57, variable: M+16

Official URL for this dataset: http://www.peptideatlas.org/PASS/PASS00254
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Servername: ftp.peptideatlas.org
Username: PASS00254
Password: AJ8939rhb

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Listing of files:

 585M Jun 10  2013 PANTUMORFINAL.sfd
 5.0K Jun 10  2013 PASS00254_DESCRIPTION-2013-05-10_105543.txt
 5.4K Jun 10  2013 PASS00254_DESCRIPTION.txt
 4.0K Jun 10  2013 PanCancer1
 4.0K Jun 10  2013 PanCancer2
 4.0K Jun 10  2013 PanCancer3
 4.0K Jun 10  2013 PanCancer5
 4.0K Jun 10  2013 PanNormal1
 4.0K Jun 10  2013 PanNormal2
 4.0K Jun 10  2013 PanNormal3
 4.0K Jun 10  2013 PanNormal5

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