Protein multiply charged

A characteristic feature of ESMS is the detection of multiply charged analytes. Macromolecules, such as proteins have multiple sites where protonation or deprotonation (the two most common charge inducing mechanisms in electrospray—other routes to charge induction include, ionization through adduct formation, through gas-phase reactions, and through electrochemical oxidation or reduction) occur. These are desorbed effectively in ESMS and... 

Multiply charged proteins can also be partially sequenced, and microsequences of proteins isolated from several microorganisms have been reported, accomplished with electrospray ionization and FTMS.23,90 Nonadjacent fragment ions may be used to identify bacterial proteins in these top-down strategies.91 In all cases these sequences could be related by bioinformatics to the parent species. An obvious extension would be to characterize proteins from intact microorganisms in this way. In at least one instance a microsequence has been obtained from a protein released from a contaminated intact bacteriophage sample (MS2) to provide a chemotaxonomic identification.77 This work was carried out in an ion trap mass spectrometer. 

The most important multiply charged polyatomic positive ions are compounds with two or more basic groups which when protonated lead to doubly or poly-charged ions. Typical examples are diamines such as the double protonated a, to alkyldiamines, H3N(CH2)pNH2+, and the most important class, the polyprotonated peptides and proteins, which have multiple basic residues. Charge reduction for these systems occurs through proton transfer from one of the protonated basic sites to a solvent molecule. Such a reaction is shown below for the monohydrate of a doubly protonated diamine ... 

The central engine of this data workflow is the process of spectral deconvolution. During spectral deconvolution, sets of multiply charged ions associated with particular proteins are reduced to a simplified spectrum representing the neutral mass forms of those proteins. Our laboratory makes use of a maximum entropy-based approach to spectral deconvolution (Ferrige et al., 1992a and b) that attempts to identify the most likely distribution of neutral masses that accounts for all data within the m/z mass spectrum. With this approach, quantitative peak intensity information is retained from the source spectrum, and meaningful intensity differences can be obtained by comparison of LC/MS runs acquired and processed under similar conditions. 

J. S. Sampson, A. M. Hawkridge, and D. C. Muddiman. Generation and Detection of Multiply-Charged Peptides and Proteins by Matrix-Assisted Laser Desorption Electrospray Ionization (MALDESI) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. J. Am. Soc. Mass Spectrom., 17(2006) 1712-1716. 

Smith, R.D. Loo, J.A. Baiinaga, C.J. Edmonds, C.G. Udseth, H.R. Colhsional Activation and Collision-Activated Dissociation of Large Multiply Charged Polypeptides and Proteins Produced hy ESI. J. Am. Soc. Mass Spectrom. 1990,1, 53-65. 

Usually the number of charges on an ion will not be known, but it can be calculated using a formula based on two different ions appearing in the spectrum. Actually, the molecular mass of a sample can be calculated automatically, or semiautomatically, by the processing software associated with the mass spectrometer. Experimentally, the automatic calculation of molecular mass is very helpful because a complex peptide or protein mixture will display an m/z spectrum with several overlapping series of multiply charged ions. 

The analysis for proteins present in plasma or a cell extract is a challenging task due to their complexity and the great difference between protein concentrations present in the sample. Simple mixtures of intact proteins can be analyzed by infusion with electrospray ionization and more complex ones by matrix assisted laser desorption ionization. MALDI is more suited for complex mixtures because for each protein an [M+H]+ signal is observed while for ESI multiply charged ions are observed. Surface enhanced laser desorption (SEEDI) is a technique for the screening of protein biomarkers based on the mass spectrometric analysis of intact proteins [49]. However in most cases for sensitivity reasons mass spec-... 

R. A., Horn, D.M., Fridriksson, E.K., Kelleher, N.L., Kruger, N.A., Lewis, M.A., Carpenter, B.K., McLafferty, F.W. Electron capture dissociation for structural characterization of multiply charged protein cations. Anal. Chem. 2000, 72, 563-73. [d] Zubarev, R.A., Haselmann, K.F., Budnik, B., Kjeldsen, F., Jensen, F. Account towards an understanding of the mechanism of electron-capture dissociation a historical perspective and modern ideas. Eur. J. Mass Spectrom. 2002, 8, 337-49. 

Figure 5 Examples of Data Generated on an Electrospray Ionization Mass Spectrometer, (a) Proteins Typically Produce Positive, Multiply Charged Ions and (b) Oligonucleotides Generate Negative, Multiply Charged Ions. Inset are the Computer-Generated Molecular Weight Spectra...

ESI tandem MS stands for electro spray ionization mass spectrometry performed in multistage. This technique is conducted based on the production of multiply charged ions from proteins and peptides. In this technique, ionization procedure is carried out within the instrument. Three types of mass analyzers are used individually or in combination. 

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