Inserito il - 26 giugno 2013 : 20:10:31
Ciao a tutti! sono nuova e trovo questo forum molto interessante.. non so se qualcuno può aiutarmi!
devo fare un'analisi critica (positiva o negativa che sia) nei confronti dell'approccio proteomico utilizzato in un articolo.. in particolare nei confronti della parte sulla spettrometria di massa.. nel lavoro sul quale stò studiando, è stata fatta un'elettroforesi 2D, sono stati tagliati gli spot e fatta una spettrometria di massa passando prima per una nano HPLC
solo che non riesco a capire bene questa diamine di spettrometria di massa, vi copio-incollo la parte dell'articolo (fa parte dei materiali e metodi) in cui parla di ciò che hanno fatto..
non saprei proprio che scriverci nelle critiche a riguardo..purtroppo internet è scarno di spiegazioni dettagliate sulla nanoHPLC accoppiata alla spettrometria di massa in tandem.. quei pochi articoli che ci sono sono bloccati e non so dove mettere le mani!!!
confido in qualcuno di voi appassionato di proteomica
ECCO LA PARTE DELL'ARTICOLO:
Protein identification with LC–MS/MS :
The gel with the highest spot intensity was selected for manual excision for evaluation by mass spectrometry. Spots of interest were carefully cut from the gel, destained in a solution containing 1.6% thiosulfate and 1% potassium ferricyanide, extensively washed in water, and then submitted to in-gel trypsin digestion. Briefly, after reduction and alkylation, trypsin digestion was performed overnight at 37 °C in 25mM ammonium bicarbonate (porcine mass spectrometry grade MSG-Trypsin; G-Biosciences, Agro-Bio, La Ferté St Aubin, France).
Peptides were extracted in 45% acetonitrile/45% water/10% tri-fluoroacetic acid (TFA) (v/v/v) and then dried in a speed-vac (Eppendorf) before nano-high pressure liquid chromatography (HPLC)–MS/MS analysis. NanoLC-NanoESI–MS/MS analyses were performed either on an ion trap mass spectrometer (LCQ Deca XP+, Thermoelectron, San Jose, CA) equipped with a nano-electrospray ion source coupled to a nano-flow high-pressure liquid chromatography system (LC Packings Dionex, Amsterdam, The Netherlands) as previously described , or on an hybrid quadrupole time-of-flight mass spectrometer (Q- Star, Applied Biosystems, Foster City, California, USA) equipped with a nano-electrospray ion source coupled with a nano HPLC system (LC Packings Dionex, Amsterdam, The Netherlands).
Peptidic samples were dissolved in 5 #956;L 95% H2O/5% ACN / 0.1% HCOOH (v/v/v) (solvent A) and were injected into the mass spectrometer using the Famos auto-sampler (LC Packings Dionex, Amsterdam, The Netherlands). Samples were desalted and concentrated on a reserved-phase precolumn of 0.3mm i.d.×5mm (Dionex) by solvent A delivered by the Switchos pumping device (LC Packings Dionex), at a flow rate of 10 #956;L/min for 3min. Peptides were then separated on a 75 #956;m i.d.×15 cm C18 Pepmap column (Dionex). The flow rate was set at 200 nL/min. Peptides were eluted using a 0% to 35% linear gradient of solvent B (25% H2O/75% ACN/0.1% HCOOH) in 80min then a 35% to 100% linear gradient of solvent B in 10min and finally 100% of solvent B was maintained for 5 min. Coated electrospray needles were obtained from New Objective (Woburn, Massachusetts, USA). The spray voltage was 1.65 kV. The mass spectrometer was operated in the positive ion mode. Data acquisition was performed in a data-dependent mode consisting of, alternatively, a full-scan MS over the range m/z 300–2000, and a full-scan MS/MS of the ion selected over the range m/z 50–2000 in an exclusion dynamic mode (themost intense ion is selected and excluded for further selection for a duration of 30 s). MS/MS data were acquired using a mass tolerance of 50mmu and the collision energy was automatically fixed by the device. For the automated data base search of fragment ion spectra, the Analyst QS software and Mascot dll script were used and final database searching was performed using Mascot software (Matrix Science London, UK, MS/MS ion search module), in the Swiss-Prot database (Sprot 0411, 525,207 sequences). Search parameters were as follows: Rattus as the taxonomic category, 100 ppm tolerance for the parent ion mass and 50mmu for the MS/MS fragment ions, one missed cleavage allowed, carbamidomethylcysteine as a fixedmodification, andmethionine oxidation as a possible modification. Only proteins with a significant Mascot score were taken into consideration and reported aftermanual verification of the fragmentation spectra.
GRAZIE A TUUUUUTTI
VI ALLEGO L'ARTICOLO CMQ
Allegato: 2012_Proteomic of hippocampus.pdf