Computational protocol: Human giant congenital melanocytic nevus exhibits potential proteomic alterations leading to melanotumorigenesis

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Protocol publication

[…] Protein separation and LC-MS analysis were performed as previously described []. Briefly, dissolved skin proteins were separated on a 12% polyacrylamide gel by SDS-PAGE. The gels were washed three times with ddH2O for 5 min each and stained with Bio-Safe Coomassie stain solution (Coomassie G250 stain; Bio-Rad, Hercules, CA) for 1 h, with gentle shaking at room temperature. The Coomassie-stained gels were evenly sliced into 15 slices and then destained by incubation in 75 mM ammonium bicarbonate/40% ethanol (1:1). Disulfides were reduced by treatment with 5 mM DTT/25 mM ammonium bicarbonate at 60°C for 30 min, followed by alkylation with 55 mM iodoacetoamide at room temperature for 30 min. The gel pieces were then dehydrated in 100% acetonitrile (ACN), dried, and swollen overnight at 37°C in 10 μl 25 mM ammonium bicarbonate buffer containing 20 μg modified sequencing-grade trypsin (Roche Applied Science, Indianapolis, IN) per ml. The tryptic peptide mixture was eluted from the gel using 0.1% formic acid. LC-MS/MS analysis was performed using a ThermoFinnigan ProteomeX workstation LTQ linear ion trap MS (Thermo Electron, San Jose, CA) equipped with a nanospray ionization (NSI) source (Thermo Electron). Briefly, 12 μl peptide sample obtained from the in-gel digestion was injected and loaded onto a peptide trap cartridge (Agilent, Palo Alto, CA). Trapped peptides were eluted onto a 10-cm reversed-phase PicoFrit column packed in-house with 5-μm, 300-Å pore size C18 and separated by gradient elution. The mobile phases consisted of H2O and ACN, both containing 0.1% v/v formic acid. The flow rate was maintained at 200 nl/min. The gradient started at 2% ACN, then reached 60% ACN in 50 min, 80% ACN in the next 5 min, and 100% H2O in the final 15 min. Data-dependent acquisition (m/z 400–1800) was enabled, and each MS survey scan was followed by five MS/MS scans within 30 s, with the dynamic exclusion option enabled. The spray voltage was 1.9 kV, the temperature of the ion transfer tube was 195°C, and the normalized collision energy was 35% [].Data-analyzed tandem mass spectra were extracted, and the charge state was deconvoluted and deisotoped using the Sorcerer 3.4 beta2 platform (Sorcerer software 3.1.4, Sorcerer Web interface 2.2.0 r334, and Trans-, Proteomic Pipeline 2.9.5). All MS/MS samples were analyzed using SEQUEST (version v.27, rev. 11; ThermoFinnigan, San Jose, CA), which was set to search the ipiHuman 3.29 database (IPI ver.3.29, 40131 entries), with semitrypsin as the digestion enzyme. The search used a fragment-ion mass tolerance of 1.00 Da and a parent-ion mass tolerance of 1.5 Da. Iodoacetamide-derivatized cysteine was specified as a fixed modification. Methionine oxidation, iodoacetamide derivatizion of cysteine, and phosphorylation of serine, threonine, and tyrosine were specified as variable modifications. The Scaffold software (version Scaffold-2.0; Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if their probability was >95.0%, as specified by the Peptide Prophet algorithm, and if they contained at least one identified peptide. Protein probabilities were assigned by the Protein Prophet algorithm. Proteins containing similar peptides such that they could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. After identifying the proteins, each dataset was used for a subtractive analysis by semi-quantitative normalized spectral counts, which were normalized by total spectral counts in the Scaffold program []. [...] A systemic bioinformatics analysis of the GCMN proteome was conducted using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING 8.3) [], the Protein Analysis Through Evolutionary Relationships classification system (PANTHER 7.0) [], the National Center for Biotechnology Information (NCBI) COG database [], Cytoscape, and ClueGO []. […]

Pipeline specifications

Software tools Comet, PANTHER, ClueGO
Databases COGs
Applications MS-based untargeted proteomics, Protein sequence analysis
Organisms Homo sapiens
Diseases Abnormalities, Drug-Induced, Breast Neoplasms, Melanoma, Neoplasms, Nevus, Pigmented