Electrospray ionization multiple charging

Following extractive deproteinization of the plasma, the amino acids (and their stable-isotope-labeled internal standards) are separated by HPLC and introduced into the mass spectrometer. Electrospray ionization results in the formation of electrically charged molecules, which are separated on the basis of their mass/charge (m/z) ratio in the first quadrupole. Following fragmentation in the collision cell, the characteristic fragment for each amino acid is selected in the second quadrupole. This process is named multiple reaction monitoring. 

Wong, S. F. Meng, C. K. Fenn, J. B. 1988. Multiple charging in electrospray ionization of poly(ethylene glycols). J. Phys. Chem., 92,546-550. 

Electrospray ionization >150,000 Ability to analyze mixtures limited sequence information for pure small peptides adapts easily for LC/MS (NP- and RP-HPLC) most suitable for quadrupoles multiple charging increases upper mass limit well suited to polar or ionic compounds Multiple charging may complicate interpretation of data glycoproteins may not yield useful information limited to flow rates <10 p.l/min... 

Data for comparison to the model were obtained via the electrospray ionization mass spectrum of a Starburst PAM AM dendrimer (core = NH3) generation = 4 between m/z 600 and 1600. The ESI mass spectrum consisted predominantly of multiple charged ions at +7, +8, +9, +10, and +11 charge states. The deconvoluted mass spectrum between 9600 and 10747 daltons suggest the presence of at least 16 components in the sample. The last significant peak at 10632 daltons corresponds with the theoretical mass 10632.96, magic number which is within one mass unit for the ideal generation = 4 structure (see Fig. 33). 

Electrospray ionization involves the introduction of a liquid solution directly into the atmospheric pressure source through an emitter. The liquid forms a droplet at the end of the emitter, where it is exposed to a high electrical field (Fig. 1). This results in a buildup of multiple charges on the surface of the droplet. The coulombic forces from these charges ultimately result in the droplet s expulsion from the surface. The ions produced in the ion source are then extracted into the mass analyzer. ESI is now widely used for identifying small molecules, proteins, studying large non-covalent complexes, structural analysis, and as a detector for separation methods such as HPLC and capillary... 

In electrospray ionization, compounds with a mass in excess of 500 Da may be prone to the formation of multiple-charge ions, either in positive-ion or in negative-ion mode [22-23], The averaging algorithm [23-24] can be used for molecular-weight determination from an ion envelope of multiple-charge ions (Ch. 16.2.2). Various automated computer-based procedures for the deconvolution or transformation of the electrospray mass spectra of proteins have been introduced. 

Coupling mass spectrometry (MS) to capillary electrophoresis provides detection and identihcation of amino acids, peptides, and proteins based on the accurate determination of their molecular masses [15]. The most critical part of coupling MS to CE is the interface technique employed to transfer the sample components from the CE capillary column into the vacuum of the MS. Electrospray ionization (ESI) is the dominant interface which allows a direct coupling under atmospheric pressure conditions. Another distinguishing features of this soft ionization technique when applied to the analysis of peptides and proteins is the generation of a series of multiple charged, intact ions. 

Electrospray ionization is classihed as a soft ionization technique. It produces molecular-weight information and very little, if any, fragmentation of the analyte ion, unless induced in the vacuum region of the mass analyzer. The number of charges accumulated by an analyte ion is proportional to its number of basic or acidic sites. The spray polarity and conditions, solution pH and nature, as well as solute concentration will all effect the charge state distribution observed in the mass spectrum. Multiple charging of an analyte ion en-... 

Figure 5 Multiple charging of a horse heart myoglobin sample analyzed by a quadrupole MS instrument with electrospray ionization collected at (a) pH 6.0 and (b) pH 3.5.

While pure electrospray nebulization is only capable of flow rates up to 20 nl/min, the development of pneumatically assisted electrospray is compatible with flow rates exceeding 200 pl/min (9). In order to successfully ionize a compound by the electrospray process the analyte should dissociate in solution to form solvated ions prior to nebulization. Ion formation from species that are not ions themselves always requires the presence of a polar atom or flmctional group to which solute cations or anions can be attached by ion-dipole forces. Biopolymers carrying multiple functional groups will form multiple charged ions, an observation that has had an outstanding impact on the analysis of peptides, proteins and oligonucleotides (7). 

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