Mass spectrometry electrospray technique

Electrospray is the softest mass spectrometry ionization technique and electrospray spectra therefore usually consist solely of molecular ions. Electrospray is unique, however, in that if the analyte contains more than one site at which protonation (in the positive-ion mode) or deprotonation (in the negative-ion mode) may occur, a number of molecular ions with a range of charge states is usually observed. For low-molecular-weight materials (< 1000 Da), the number of sites... 

With the advent of electrospray mass spectrometry and the re-emergence of time of flight mass spectrometry, the technique will be of major use in the quality control of therapeutic antibodies and peptides. 

Electrospray in the mid 1980s revolutionized biological mass spectrometry, in particular in the field of protein and peptide sequence analysis. Electrospray is a concentration-dependent, rather than a mass-dependent process, and maximum sensitivity is achieved at low flow rates with high-concentration, low-volume samples (Griffiths 2000). Joint NMR, x-ray diffraction, electrophoresis, and chromatography techniques with mass spectrometry (MS) techniques would be a trend in the future. 

The two mainstays of modem mass spectrometry, electrospray MS and MALDI/ TOFMS, can both be used with adequate sensitivity for single-bead detection of molecular weight and structural information [37], Single-bead MS using TFA cleavage and MALDI/ TOF analysis is now a well-established technique [38 11], A Scripps group [42] reports... 

The detection and identification of phenolic compounds, including phenolic acids, have also been simph-fied using mass spectrometry (MS) techniques on-hne, coupled to the HPLC equipment. The electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) interfaces dominate the analysis of phenohcs in herbs, fmits, vegetables, peels, seeds, and other plants. In some cases, HPLC, with different sensitivity detectors (UV, electrochemical, fluorescence), and HPLC-MS are simultaneously used for the identification and determination of phenolic acids in natural plants and related food products.In some papers, other spectroscopic instmmental techniques (IR, H NMR, and C NMR) have also been apphed for the identification of isolated phenolic compounds. 

S. Hillenkamp, F De Vries, M.S. Comparative Mass Spectrometric Analyses of Photofrin Oligomers by Fast Atom Bombardment Mass Spectrometry, UV and IR Matrix-Assisted Laser Desorption/lonization Mass Spectrometry, Electrospray Ionization Mass Spectrometry and Laser Desorption/Jet-Cooling Photoionization Mass Spectrometry, J. Mass Spectrom. 34, 661-669 (1999). Powell, K.D. Fitzgerald, M.C. Accuracy and Precision of a New H/D Exchange- and Mass Spectrometry-Based Technique for Measuring the Thermodynamic Properties of Protein-Peptide Complexes, Biochemistry 42,4962-4970 (2003). 

This section will cover the two most popular ionization techniques for analyzing proteins by mass spectrometry electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). A brief description of five common mass spectrometers that have been coupled to these ion sources will follow. 

X-Ray Absorption Spectroscopy Applications of Electrospray Mass Spectrometry Other Techniques... 

Banerjee, S., Mazumdar, S. (2012) Electrospray Ionization Mass Spectrometry A Technique to Access the Information beyond the Molecular Weight of the Analyte. Int. J. Anal. Chem. 2012 282574. 

Fast atom bombardment, liquid-SIMS (secondary ion mass spectrometry), electrospray (ESI), and matrix assisted laser desorption (MALDI) ionization modes have been applied successfully for the investigations of biomolecules.However, ESI and MALDI are the two most frequently adopted techniques for investigations of biopolymersDetails involving the principles and application of all of these techniques can be found elsewhere. The samples may be introduced either directly or after liquid chromatographic separation. All of the above techniques, with the exception of MALDI, have been adopted for the LC/MS experiments. " Although most of the reported LC/MS investigations involved the electrospray ionization of the molecules, continuous flow-FAB ionization techniques have also been found useful. 

Another type of ion is formed almost uniquely by the electrospray inlet/ion source which makes this technique so valuable for examining substances such as proteins that have large relative molecular mass. Measurement of m/z ratios usually gives a direct measure of mass for most mass spectrometry because z = 1 and so m/z = m/1 = m. Values of z greater than one are unusual. However, for electrospray, values of z greater than one (often much greater), are quite coimnonplace. For example, instead of the [M + H]+ ions common in simple Cl, ions in electrospray can be [M + n-H]- where n can be anything from 1 to about 30. 

However, interpretation of, or even obtaining, the mass spectrum of a peptide can be difficult, and many techniques have been introduced to overcome such difficulties. These techniques include modifying the side chains in the peptide and protecting the N- and C-terminals by special groups. Despite many advances made by these approaches, it is not always easy to read the sequence from the mass spectrum because some amide bond cleavages are less easy than others and give little information. To overcome this problem, tandem mass spectrometry has been applied to this dry approach to peptide sequencing with considerable success. Further, electrospray ionization has been used to determine the molecular masses of proteins and peptides with unprecedented accuracy. 

Two relatively new techniques, matrix assisted laser desorption ionization-lime of flight mass spectrometry (MALDI-TOF) and electrospray ionization (FS1), offer new possibilities for analysis of polymers with molecular weights in the tens of thousands. PS molecular weights as high as 1.5 million have been determined by MALDI-TOF. Recent reviews on the application of these techniques to synthetic polymers include those by Ilantoif54 and Nielen.555 The methods have been much used to provide evidence for initiation and termination mechanisms in various forms of living and controlled radical polymerization.550 Some examples of the application of MALDI-TOF and ESI in end group determination are provided in Table 3.12. The table is not intended to be a comprehensive survey. 

For many years, electron ionization, then more usually known as electron impact, was the only ionization method used in analytical mass spectrometry and the spectra encountered showed exclusively the positively charged species produced during this process. Electron ionization also produces negatively charged ions although these are not usually of interest as they have almost no structural significance. Other ionization techniques, such as Cl, FAB, thermospray, electrospray and APCI, however, can be made to yield negative ions which are of analytical utility. 

Electrospray ionization occurs by the same four steps as listed above for thermospray (see Section 4.6). In contrast to thermospray, and most other ionization methods nsed in mass spectrometry, it shonld be noted that electrospray ionization nnnsnally takes place at atmospheric pressure. A similar process carried out under vacuum is known as electrohydrodynamic ionization and gives rise to qnite different analytical results. This technique has not been developed into a commercial LC-MS interface and will not be considered further. 

Electrospray is an unusual mass spectrometry technique in that it allows the study of the three-dimensional structure of compounds, particularly proteins, in solution as it is believed that this is relatively unchanged when ions are transferred to the vapour phase. This type of application will be discussed in more detail in Chapter 5 but attention is drawn at this point to the previous comments regarding the effect that the HPLC conditions, such as pH, may have on the appearance of an electrospray spectrum and the conformational deductions that may be made from them. 

MS-MS is a term that covers a number of techniques in which two stages of mass spectrometry are used to investigate the relationship between ions found in a mass spectrum. In particular, the product-ion scan is used to derive structural information from a molecular ion generated by a soft ionization technique such as electrospray and, as such, is an alternative to CVF. The advantage of the product-ion scan over CVF is that it allows a specific ion to be selected and its fragmentation to be studied in isolation, while CVF bring about the fragmentation of all species in the ion source and this may hinder interpretation of the data obtained. 

Snyder, A.P., Electrospray a popnlar ionization technique for mass spectrometry, in Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry, ACS Symposium Series, Snyder, A.P. and Anaheim, C.A., Eds., Washington, D.C., 1995, chap. 1. 

LC/MS/MS. LC/MS/MS is used for separation and quantitation of the metabolites. Using multiple reaction monitoring (MRM) in the negative ion electrospray ionization (ESI) mode, LC/MS/MS gives superior specificity and sensitivity to conventional liquid chromatography/mass spectrometry (LC/MS) techniques. The improved specificity eliminates interferences typically found in LC/MS or liquid chro-matography/ultraviolet (LC/UV) analyses. Data acquisition is accomplished with a data system that provides complete instmment control of the mass spectrometer. 

On the other hand, electrospray ionization mass spectrometry (ESMS) has been first combined with electrochemistry at ITIES in order to confirm the stoichiometry of a complex ion transferred into an organic phase directly. ESMS is now becoming a popular and powerful technique not only in chemistry but also in biology, pharmacy, medical science, etc. Electrospray (ES) ionization is exceedingly effective resource for producing gas-phase ions from various solutions which contain any kinds of ion. Thus, ESMS can sometimes give us highly useful information in comparison with electrochemical results. 

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