Project PXD005274

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Proteomic survey for search of biomarkers of colon tumors


To the search of new colon tumor biomarkers in the transition from normal colon (NC) mucosa to adenoma (AD) and adenocarcinoma (AC), we integrated microarray data with the results of a high-throughput proteomic workflow. In proteomic study, we used a modified isoelectric focusing protocol on strips with an immobilized pH gradient to separate peptides labeled with iTRAQ (isobaric tags for relative and absolute quantitation) tags followed by liquid chromatography–tandem mass spectrometry analysis.

Sample Processing Protocol

Proteins were precipitated from equal amounts (by protein content) of samples of each subcellular fraction from each tissue type using the ProteoExtract Protein Precipitation Kit (Calbiochem) according to the manufacture r’s protocol. The combined samples were prepared by mixing together equal protein amounts of four subcellular aliquots from an individual tissue sample followed by precipitation step. Protein pellets were resuspended in 200 μl of dissolution buffer (DB; 0.5 M triethylammonium bicarbonate with 0.1% SDS; both reagents were provided with the iTRAQ kit from Applied Biosystems). To facilitate protein solubilization, samples were vortexed thoroughly and (optionally) treated with a 500W Cole-Parmer ultrasonic homogenizer (amp. 24%, pulse 2 s, gap 2 s; 5 pulses). The protein concentrations of the combined and subcellular samples were measured by the Bradford method. Aliquots of samples (100 μg) were stored at -72 °C. Before labeling, protein aliquots were evaporated to dryness in a speedvac, dissolved in 30 μl DB, reduced, cysteine-blocked, and digested overnight with trypsin (Promega). The combined and subcellular samples of normal colonic mucosa, tubulo-villous adenoma, tubular adenoma, and adenocarcinoma were differentially labeled with one of the four iTRAQ tags (114, 115, 116, and 117, respectively) for 1 h as per the protocol from the manufacturer (Applied Biosystems). After 1 h of iTRAQ tagging, the reaction was quenched for 30 min by adding 100 μl H2O. iTRAQ-labeled samples were combined and resuspended in 700 μl buffer [8 M urea plus 2% IPG buffer pH 3.5–5 (GE Healthcare) in 50 mM Tris-HCl, pH 8.0]. The solution was divided and applied to two 18-cm IPG strips with 3.5–4.5 pH gradients (GE Healthcare): 350 μl of sample/strip, the equivalent of 200 μg protein. The IPG strips were covered with mineral oil and rehydrated overnight. The next day, the strips were isoelectrofocused (IEF) using a Multiphor II electrophoresis system (GE Healthcare) as follows. First, there was a 5-h pre-run at 200 V during which the paper electrodes were changed every hour. Second, a standard gradient focusing program was used: 0.1 h at 500 V, 1.5 h at 3500 V, and 15.5 h at 3500 V. After focusing, the strips were removed from the tray and the overlay oil was blotted with a paper tissue. Strips were stored at -72°C in a glass tubes. The strips were placed next to each other on a cold tray on dry ice and cut simultaneously into 5mm pieces wit h a razor blade. The paired pieces from the two strips were transferred into individual 1.5-ml Eppendorf tubes. Finally, the 18-cm long gel strips were sliced into 35 sections. Peptides were extracted three times from each section by adding 150 μL 0.1% TFA/2% acetonitrile to each section and vortexing the tubes for 20 min at room temperature. The combined extracts (450 μl) were then overlaid two times with 200-μl hexane. Each time the tube was mixed for 15 s; after phase separation, the hexane layer was discarded. The remaining extracts were evaporated to dryness by vacuum centrifugation, the pellets were dissolved in 70 μl 0.1% formic acid, and 20-μl aliquots were stored at -72 °C for LC-MS/MS analysis. The peptide mixture was applied to the nanoACQUITY UPLC Trapping Column (Waters) using water containing 0.1% formic acid as the mobile phase and then transferred to the nanoACQUITY UPLC BEH C18 Column (Waters, 75 μm inner diameter; 250-mm long) using an acetonitrile gradient (3–33% acetonitrile over 150 minutes) in the presence of 0.05% formic acid with a flow rate of 250 nl/min. The column outlet was directly coupled to the ion source of the Q-Tof Premier tandem mass spectrometer (Waters) working in the regime of data-dependent MS to MS/MS switch. A blank run to ensure a lack of cross contamination from previous samples preceded each analysis.

Data Processing Protocol

MS raw data files were processed to peak lists wit h the Mascot Distiller software (version 2.2.1, Matrix Science, London, UK). The output list of precursor and product ions was compared to the Swiss-Prot database with the taxonomy restricted to Homo sapiens using the MASCOT search engine (version 2.2.03, Matrix Science, London, UK). The search parameters were set as follows: enzyme, semi-trypsin; fixed modification, cysteine modification by MMTS as well as iTRAQ labeling of the N-terminus of peptides and of lysine side chains; variable modifications, oxidation (M), phosphorylation of serine, threonine (ST), and tyrosine (Y); protein mass, unrestricted; max missed cleavages, 1.


Michał Kistowski, Institute of Biochemistry and Biophysics PAS
Michał Dadlez, Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics PAS ( lab head )

Submission Date


Publication Date




Cell Type

epithelial cell


colon cancer


Q-Tof Premier


Not available



Experiment Type

Shotgun proteomics

Assay count



    Mikula M, Rubel T, Karczmarski J, Goryca K, Dadlez M, Ostrowski J; Integrating proteomic and transcriptomic high-throughput surveys for search of new biomarkers of colon tumors., Funct Integr Genomics, 2011 Jun, 11, 2, 215-24, PubMed: 21061036

    Kistowski M, Dębski J, Karczmarski J, Paziewska A, Olędzki J, Mikula M, Ostrowski J, Dadlez M. A strong neutrophil elastase proteolytic fingerprint marks the carcinoma tumor proteome. Mol Cell Proteomics. 2016 Dec 7. pii: mcp.M116.058818 PubMed: 27927741


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# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 70277 80215mik_5c2.xml 21 30 22 194 0
2 70398 80403mik3_8a.xml 211 657 502 1564 0
3 70276 80310mik4_6.xml 57 179 108 609 0
4 70397 80326mik3_18a.xml 464 1839 1417 2345 0
5 70279 80710mik2b_30.xml 155 413 348 1434 0
6 70278 80404mik2_3a.xml 112 327 267 1670 0
7 70399 80605mik1B_7.xml 122 272 223 897 0
8 70273 80218mik_22c2.xml 467 2373 1632 2968 0
9 70394 80218mik_23c2.xml 412 1981 1449 3072 0
10 70272 80710mik2b_28.xml 242 644 512 1633 0