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Tag! You’re a phosphopeptide The raison d’être of proteomics is cataloging the molecular changes that stem from development, mutation, or exposure to environmental or chemical forces. Often, though, the proteins themselves do not change as much as their chemical adornments do. Posttranslational modifications such as phosphorylation, acetylation, and glycosylation can alter a protein’s localization, activity, or stability. As a result, protein chemists typically are very interested in documenting these chemical changes. Yet MS of modified peptides is no trivial matter. For one, these peptides represent only a relatively small fraction, so a thorough analysis requires enrichment. Moreover, modified peptides present particular MS challenges, because they are less amenable to sequencing by collision-induced dissociation and may not ionize as efficiently as unmodified peptides. In research published in AC (2008, 80, 2531–2538), Hubert Girault of the École Polytechnique Fédérale de Lausanne (Switzerland) and colleagues address the second issue for phosphorylated peptides. The team coupled a previously described tagging chemistry with a novel microfluidic ESI source to label phosphopeptides during MS injection itself. “The idea here was to couple our knowledge in microfabrication and electrochemistry to tag specific molecules, in this case, phosphopeptides,” says graduate student and lead author Michel Prudent. The approach serves a dual function, Prudent notes. First, it induces a 581 Da mass shift of phosphorylated peptides, thereby marking them in the resulting spectra. It also should boost the peptides’ ionization efficiency by making them more positively charged. The key to this process is 1,3-bis [bis(2-pyridylmethyl)amino]-2-propanol (LH). In the presence of Zn(II) cations, LH forms 1,3-bis[bis(2pyridylmethyl]amino]-propan-2-olato
Pt electrode + Zn2L(OAc)(PF6)2
Phosphopeptides
or Zn electrode + LH
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A dual-channel microfluidic ESI source tags phosphopeptides for MS analysis.
dizinc(II)2+, which can coordinate a peptide phosphate group (ROPO32–) and serves as a kind of tag. Previous researchers already had demonstrated that this tag could capture and mark phosphopeptides for MALDI MS analysis; Girault and his team, however, applied it to electrospray analysis. In a first trial, the team synthesized the tag compound off-line. They added it to a simple mixture of four phosphorylated and unmodified synthetic peptides and used a standard electrospray interface to inject the sample into a mass spectrometer. As expected, phosphorylated peptides were observed in both untagged and tagged forms, whereas unmodified peptides remained unlabeled. Some contamination with unreacted tag and its PF6 – counterions also was observed. The team then switched to a dualchannel microfluidic device custombuilt for this study. One channel contained the peptide sample, the other the tag compound. The driving pressure came from the weight of the reagent liquids in their accompanying reservoirs, and an on-chip electrode provided the current for the ionization itself. The team first loaded the two channels with peptide mixture and tag compound (previously synthesized), allowing the two reagents to mix at the tip of the chip, in the Taylor cone. When
a platinum electrode was used to induce ionization, the results were similar to those with a standard electrospray interface. Later, Girault’s team realized that they could prepare the tag compound on the fly by using a sacrificial zinc electrode instead of platinum and by allowing the LH to react with the Zn(II) that would arise by oxidation of the zinc electrode. “We used this intrinsic electrochemical property to produce zinc cations by oxidation directly on the chip, and that reacts with our ligand to produce our tag,” says Prudent. “This tag then reacts within the Taylor cone with the phosphocompound.” The data mirrored those obtained with previously synthesized tag reagent. But generation of the tag doesn’t happen immediately, Prudent says; there is a delay of several minutes as Zn(II) accumulates. Tagging efficiency fluctuated with time, current, and concentrations of LH and the peptide. At 10 μM LH, the conversion rate of a phosphorylated angiotensin II peptide to its tagged derivative reached ~0.4 after 45 minutes. Conversion could be accelerated, however, by decreasing the concentration of the peptide. According to Prudent, the group has several options for optimizing this technique, including tweaking reagent concentrations, flow rate, and chip design. More significantly, he says he is unsure how the technique will perform on a complex biological sample, such as serum or plasma. For those interested in trying the approach, Prudent says it will work with any ESI-based mass spectrometer, although it requires some instrumental modification. The existing ESI interface must be removed and replaced with some way to hold, position, and control the chip. The chip, though, is an offthe-shelf product, available from DiagnoSwiss SA. a —Jeffrey M. Perkel
A p r i l 1 , 2 0 0 8 / A n a ly t i c a l C h e m i s t r y
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