2D LC : différence Offline vs Online
Bonjour,
Je suis sur un article de protéomique, et ils parlent de 2D LC Offline et Online, je vois pas la différence, est-ce quelqu'un saurait ?
J'ai cherché sur internet ou d'autres publis, mais c'est pas clair : Flux en continue pour la online ou bien ca serait une histoire de concentration de sels pour éluer l'échangeuse de cations SCX...
merci
Bonjour,
Tu aurais le passage voire même la référence de ton article s'il te plaît ? Ca sera plus simple pour voir de quoi il s'agit exactement.
Dans les articles sur les méthodes d'études des protéines par des systèmes complexes comme LC-ESI-MS/MS, il n'est pas rare que les auteurs couplent leur système à des bases de données en ligne dédiées à des spectres de masse peptidiques. Ainsi, grâce à ce couplage, l'analyse est entièrement automatisée jusqu'au résultat final puisque l'ordinateur collecte le spectre de masse, se connecte à la base de données, compare le profil de fragmentation avec les profils préexistant et identifie la protéine.
Il y a donc fort à parier que offline et online soit l'absence ou la présence, respectivement, de connexion du système à internet.
Greg
Merci pour la réponse,
voici l'article en question :
Discovery and Verification of Head-and-neck Cancer Biomarkers by Differential Protein Expression Analysis Using iTRAQ Labeling, Multidimensional Liquid Chromatography, and Tandem Mass Spectrometry.
Molecular & Cellular Proteomics 7:1162–1173, 2008.
Et le passage en question :
Strong Cation Exchange (SCX) Separation Conditions—For the off-line 2D LC-MS/MS analysis, each set of labeled samples was first separated by SCX fractionation using an HP1050 high performance liquid chromatograph (Agilent, Palo Alto, CA) with a 2.1-mm-internal diameter 100-mm-length Polysulfoethyl A column packed with 5-m beads with 300-Å pores (The Nest Group, Southborough, MA) as described previously (17). A 2.1-mm-internal diameter 10-mmlength guard column of the same material was fitted immediately
upstream of the analytical column. Separation was performed as described previously (17). Briefly each pooled sample set was diluted with the loading buffer (15 mM KH2PO4 in 25% acetonitrile, pH 3.0) to a total volume of 2 ml, and the pH was adjusted to 3.0 with phosphoric acid. Samples were then filtered using a 0.45-m syringe filter (Millipore, Cambridge, Ontario, Canada) before loading onto the column. Separation was performed using a linear binary gradient over 1 h. Buffer A was identical in composition to the loading buffer; Buffer B was Buffer A containing 350 mM KCl. Fractions were collected every
2 min using an SF-2120 Super Fraction Collector (Advantec MFS, Dublin, CA) after an initial wait of 2 min to accommodate the void volume. This resulted in a total of 30 SCX fractions per sample set. These fractions were dried by speed vacuuming (Thermo Savant SC110 A, Holbrook, NY) and resuspended in 30 l of 0.1% formic acid each.
For the on-line 2D LC-MS/MS analysis, an SCX cartridge (BioXSCX, LC Packings, Amsterdam, The Netherlands) was plumbed upstream of the reverse phase (RP) desalting cartridge and analytical column. This SCX cartridge was connected through a second valve on the Switchos unit as shown in Fig. 1. Samples were separated on this SCX cartridge using 10-l step elutions with increasing concentration of ammonium acetate (10 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 500 mM, and 1 M). Each step elution was loaded onto the RP desalting column using the switching program
as shown in Fig. 1 where the eluting peptides were desalted before loading onto the analytical column that was subsequently brought in line with the desalting column. The flow path used for these steps was designed to ensure that there was never any flow reversal through either of the cartridges (SCX or RP). Separation on the RP analytical column was effected as described for the second stage of the off-line LC-MS/MS analysis described below.
