Peptides ionization

JENSEN, O.N., PODTELEJNKOV, A., MATTHIAS-MANN, M., Delayed extraction improves specificity in database searches by matrix-assisted laser desorption/ionization peptide maps, Rapid Comm. Mass Spectrom., 1996,10, 1371-1378. 

FIGURE 15.2 Common protein ionization methods used for MS-based proteomics. Two common ionization technologies are currently available for protein analysis. Top ESI volatilizes and ionizes peptides and proteins in solution. Bottom MALDI uses analytes that are co-crystallized in a matrix composed of organic acid on a solid support. A pulse of ultraviolet laser evaporates the matrix and analyte into gas phase, resulting in generation of single charge ions. 

Mann, M. and Talbo, C. (1996) Developments in matrix-assisted laser desorption ionization peptide mass spectrometry. Current Opinion in Biotechnology 7, 11-1 9. 

They screened seven libraries, each containing 19 peptides of different masses, and found the sequences that bound most tightly to Tom20. Matrix-assisted laser desorp-tion/ionization time-of-flight mass spectrometry (MALDI-TOF MS) served as a readout that allowed the ionized peptides to be observed directly. Using this approach, Kohda and colleagues discovered that the recognition site spans six residues, not the previously established five, and were able to refine sequence preferences further. 

Mass spectrometers consist of three components an ionization source, a mass analyzer, and a detector. First, the ionization source adds charge to the proteins or peptides in the sample, typically in the form of a proton to produce positively charged particles, and injects them into a vacuum chamber. Second, a mass analyzer uses an electromagnetic held to separate and sort the ionized peptides. Third, a detector registers the number of ions at each mass-to-charge value. The two most technically demanding components are the ionization sources, which we have discussed in the previous section, and the mass analyzers. 

In a series of studies we recently demonstrated (29, 30, 63-67) that the resolution of peptides on reversed phase can be profoundly influenced by the addition of appropriate counterionic reagents to a mobile phase of deflned pH, ionic strength, and water content. Retention under these conditions can be discussed in terms of ion-air associations between the ionized peptide and a counterion in the mobile phase and subsequent sorption of the complex onto the stationary phase. Alternatively, adsorption of the counterion, particularly if it is lipophilic, onto the nonpolar stationary phase may occur, and peptide retention would then be mediated by dynamic liquid-liquid ion-exchange effects. Arguments in favor of the participation of one, the other, or both of these alternative pairing-ion phenomena in ion-pair chromatography have been extensively reviewed (16, 28b, 62, 68, 68a). It can be shown (62, 68) that retention behavior in ion-pair systems can be described by... 

Since pH conditions can be readily chosen to ensure that peptides are significantly ionized, peptide elution orders from nonpolar phases with ion-pairing elution systems will be determined by their relative hydro-phobicities and the polarity or charge density of the counterion. Major changes in selectivity and retention times of peptides on reversed-phase... 

Both IR and UV/vis action spectra were used [139] to characterize the radical cations of Trp-containing peptides, in particular AcGly3TrpNH2. Both types of spectra indicated a canonical n-radical cation structure, ruling out the possibilities of forming a zwitterion, or of an ionization site other than the indole jt-system. The spectra are shown in Fig. 15 (compared with the IR spectrum of the protonated peptide). These spectroscopic probes are seen in general as useful structural diagnostics for ionized peptides containing a Tip residue. 

The whole trick to success with MALDI is finding the right matrix. Some matrices are extremely good at ionizing peptides, others are preferred for proteins, others for DNA, still others for oligosaccharides. In addition to Chapter 7 in this volume, there s a large but... 

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