Electrospray protein analysis

Table 5.8 Polypeptides detected during the LC-electrospray-MS analysis of the tryptic digest from / -lactoglobulin (/ILG). Reprinted from 7. Chromatogr., A, 763, Tnrnla, V. E., Bishop, R. T., Ricker, R. D. and de Haseth, J. A., Complete structnre elncidation of a globular protein by particle beam hqnid chromatography-Fourier transform infrared spectrometry and electrospray hqnid chromatography-mass spectrometry - Seqnence and conformation of / -lactoglobulin , 91-103, Copyright (1997), with permission from Elsevier Science...

Bolton, J. L. Le Blanc, J. C. Y. Siu, K. W. M. Reaction of quinone methides with proteins analysis of myoglobin adduct formation by electrospray mass spectrometry. Biol. Mass... 

Zhang, B., Foret, F, and Karger, B. L. (2000). A microdevice with integrated liquid junction for facile peptide and protein analysis by capillary electrophoresis/electrospray mass spectrometry. Anal. Chem. 72, 1015-1022. 

Characterization of noncovalent bonding of the proteins can also be done using MS. For example MALDI MS has been used in measurement of the molecular mass of the noncovalendy linked tetramer of glucose isomerase, a complex consisting of identical monomers of 43.1 kDa each. MALDI-TOFF peptide mass fingerprinting combined with electrospray tandem mass spectrometry can efficiently solve many complicated peptide protein analysis problems. 

Q. Tang, A.K. Harrata and C.S. Lee, Capillary isoelectric-focusing electrospray mass-spectrometry for protein analysis. Analytical Chemistry, 67, 3515-3519 (1995). 

Recent advances in protein analysis by MS are due to the introduction of electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI), MSN scan modes, as well as improvements in instrument sensitivity, resolution, and mass accuracy. With these improved techniques, researchers will continue to use MS to help elucidate primary, secondary, and to a lesser extent, tertiary structure of proteins. 

Balaguer E, Demelbauer U, Pelzing M, Sanz-Nebot V, Barbosa J, Neususs C. Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis— electrospray—time-of-flight mass spectrometry. Electrophoresis 2006 27 2638-50. 

After the first demonstration of multiply charged gas-phase proteins ions, all major instrument manufacturers developed atmospheric-pressure ion sources, equipped with electrospray interfaces for both protein characterization and LC-MS applications. Within 5 years, electrospray interfacing became the method of choice in LC-MS coupling. It led to a large increase in the use of MS for the characterization and identification of labile and polar analytes as well as to routine quantitative analysis. The advent of electrospray ionization for peptide and protein analysis stimulated further development and analytical application of existing and new mass analysis approaches, such as quadrupole ion traps, Fourier-transform ion-cyclotron resonance MS, and quadru-pole-time-of-flight hybrid instruments. It opened new application areas, such proteomics. LC-MS... 

Shieh, I.F. Lee, C.Y. Shiea, J. Eliminating the Interferences from TRIS Buffo and SDS in Protein Analysis by Fused-Droplet Electrospray Ionization Mass Spectrometry. J. Proteome Res. 2005, 4, 606-612. 

Liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) was introduced in the 1980s [1]. Today it has become a standard method for separation and characterization of nonvolatile compounds. Reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to ESI-MS is the method of choice for peptide and protein analysis, but also used for the characterization of contaminants, therapeutic drugs, and food additives [2-5], More than 75% of HPLC analyses are run on RP stationary phases, and a wide range of columns are available with various substituents of the silica matrix, base deactivation, endcapping, and column dimensions. 

Loo, J.A., Udseth, H.R., and Smith, R.D. (1989) Peptide and protein analysis by electrospray ionization-mass spectrometry and capillary electrophoresis-mass spectrometry. Analytical Biochemistry, 179,404-412. 

For mixture.s the picture is different. Unless the mixture is to be examined by MS/MS methods, usually it will be necessary to separate it into its individual components. This separation is most often done by gas or liquid chromatography. In the latter, small quantities of emerging mixture components dissolved in elution solvent would be laborious to deal with if each component had to be first isolated by evaporation of solvent before its introduction into the mass spectrometer. In such circumstances, the direct introduction, removal of solvent, and ionization provided by electrospray is a boon and puts LC/MS on a level with GC/MS for mixture analysis. Further, GC is normally concerned with volatile, relatively low-molecular-weight compounds and is of little or no use for the many polar, water soluble, high-molecular-mass substances such as the peptides, proteins, carbohydrates, nucleotides, and similar substances found in biological systems. LC/MS with an electrospray interface is frequently used in biochemical research and medical analysis. 

A typical electrospray analysis can be completed in 15 min with as little as 1 pmol of protein. An analysis of the cord blood of a baby (Figure 40.6) showed quite clearly that five globins were present, viz., the normal ones (a, (3, Gy, and Ay) and a sickle-cell variant (sickle (3). The last one is easily revealed in the mass spectrum, even at a level of only 4% in the blood analyzed. 

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