WORKING WITH SERA KNOWS WELL THAT ALBUMIN OBLITERATESTHE SIGNAL OF...

10-

working with sera knows well that albumin obliteratesthe signal of most proteins co-focusing in the same pIregion. It just so happens that human serum albumin

Fig. 5.

Analysis of monoclonal Igs from mouse hybridomas, purified

focuses in the pH 5–6 region (under denaturing condi-

by

mercapto-ethyl-pyridine–HyperCel

chromatography,

via

two-

tions), because of a multitude of isoforms [33]. Thus, all

dimensional maps. (A) Control monoclonal antibodies (untreated);

proteins that focus in this region have to fight against

(B) monoclonal antibodies after Equalizer Bead treatment. Newly

this ‘Goliath’ for survival. From this point of view, as

revealed spots eluted and analysed by liquid chromatography-

MS

⁄

MS. Staining with Sypro Ruby. First dimension: nonlinear

Equalizer Beads are not meant to select a single protein,

pH 3–10 immobilized pH gradients. Second dimension: SDS

⁄

PAGE

such as antibodies, or protein family, such as lectins, or

in a 8–10% polyacrylamide gel slab.

to capture specific components, but rather to embraceall proteins in a proteome, they are ecumenical (or atdepending on the amount of extract loaded and theleast they try to be), i.e. they accept and adopt all‘faiths, colours, races and creeds’. In addition, theybead volume.Analysis of purification may also have utility duringintroduce ‘democracy’ in a rather ‘oligarchic’ (somewould say ‘plutocratic’) proteome. Another majorthe development of second-generation processes for agiven biopharmaceutical, where demonstration of com-bonus of the approach described is the capture andparability is to be made not only for the degree of pur-adsorption of a high proportion of small and large pep-tides (in the 600–8000-Da range) that are normally lostity of the target protein but also for the qualitativeupon two-dimensional electrophoretic mapping. Such aand quantitative presence of traces of impurity.peptide population in human sera may be of particularEqualizer Beads can be applied here for two proces-ses: in the first instance, for tracking and concentratingimportance as it may contain protein cleavage productssuch impurities, so as to render them amenable toof diagnostic value [34].identification by MS and other analytical techniquesThere is at least one major limitation to the present(in this case, a small amount of beads is incubatedmethod: owing to the fact that the interaction mechan-with large sample volumes and quantities); by the sameism is rather delicate (it encompasses all types of bondsthat help to stabilize the tridimensional structure ofand S.cerevisiae extract (Dr M. Toledano and Dr N.proteins, such as ionic, hydrogen bonding and hydro-le Moan, CEA Saclay, France), as well as providers ofexperimental data (Dr V. Thulasiraman, Dr L. Guer-phobic association, and other weak interactions suchrier, Dr F. Fortis, Dr P. Antonioli).as van der Waals forces) adsorption can only beobtained under native physiological conditions, i.e. inthe absence of strong denaturants. Thus, membraneReferencesand very hydrophobic proteins, which normally requirea strong solubilizing agent for dissolution, cannot be1 Conan Doyle A. (2005)The Casebook of Sherlock Hol-mes. BBC Consumer Publishing, London.recovered, as mild conditions are required when Equal-2 Anderson NL & Anderson NG (2002) The humanizer Beads are incubated with any proteome; for exam-plasma proteome: history, character and diagnostic pro-ple, TUC (thiourea, urea and CHAPS solubilizingspects.Mol Cell Proteomics1, 845–867.solution), a classical solubilizing cocktail in two-dimen-3 Anderson LN, Polanski M, Pieper R, Gatlin T, Tiruma-sional maps, is typically used for desorption of pro-lai RS, Conrads TP, Veenstra TD, Adkins JN, Poundsteins bound to the beads.JG, Fagan R,et al.(2004) The human plasma proteome.Another matter of concern regards the possibility ofMol Cell Proteomics3, 311–326.abnormal binding of proteins to the beads, leading to4 Christie A (1934) Murder on the Orient Express. Arche-unequal situations. It is unrealistic to think that alltte, Paris.proteins will behave well towards the adsorbing ligand5 Edwards B (1963)The Pink Panther. Paramount,library. Working with sera, we have found at least oneHollywood, CA.protein with unexpected behaviour: apolipoprotein J6 Bae SH, Harris AG, Hains PG, Chen H, Garfin DE,(Apo J), which is greatly enriched compared with allHazell SL, Paik YK, Walsh B & Cordwell SJ (2003)other serum components, rendering it the most abun-Strategies for the enrichment and identification of basicdant component after equalization. Apo J possesses aproteins in proteome projects.Proteomics3, 569–579.large number of binding sites for several components,7 Pedersen SK, Harry JL, Sebastian L, Baker J, Trainisuggesting that it may recognize more than one hexa-MD, McCarthy JT, Manoharan A, Wilkins MR, Goo-peptide ligand, thus saturating an abnormal number ofley AA,et al.(2003) Unseen proteome: mining belowsites in a larger bead population compared with otherthe tip of the iceberg to find low abundance and mem-‘well-behaved’ proteins. Close examination of two-brane proteins.J Proteome Res2, 303–312.dimensional maps suggests that a few other proteins8 Righetti PG, Castagna A, Herbert B, Reymond F &might exhibit similar behaviour, although to whatRossier JS (2003) Prefractionation techniques in pro-extent this abnormal behaviour will affect the totalteome analysis.Proteomics3, 1397–1407.9 Righetti PG, Castagna A, Antonioli P & Boschetti Eproteome of a tissue is yet to be investigated.(2005) Prefractionation techniques in proteome analysis:the mining tools of the third millennium.ElectrophoresisConclusions