Electrospray-Time of Flight-Mass Spectrometry

Online LC-ESI-TOF-MS experiments are carried out in a very similar fashion to the off-line NPS-HPLC separations described above, with a few notable exceptions. Firstly, 0.3% (v/v) formic acid is added to each mobile phase to counteract the ionization suppression induced by TFA. Because of the formic acid UV detection must be carried out at 280 nm (as opposed to 214 nm). To aid in normalization between runs 1 jag of Bovine insulin (MW = 5734 Da) is added to each chromatofocusing fraction prior to injection onto the column. Finally, the flow is split postcolumn directing 200 JlL/min into the ion source and the remaining 300 JlL/min through the UV detector and fraction collection. 

Because online separations provide such a wealth of information about target proteins, interpretation becomes of critical importance in order to make full use of the data. The first step in any analysis of LC-MS data involves integration and deconvolution of sample spectra to determine protein mass and intensity. In manual analysis (Hamler et al., 2004), users identify protein umbrellas, create a total ion chromatogram (TIC), integrate the protein peak, and deconvolute the resulting spectrum. Deconvolution of ESI spectra employs a maximum entropy deconvolution algorithm often referred to as MaxEnt (Ferrige et al., 1991). MaxEnt calculates 

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Banks, J. F., Gulcicek, E. E. (1997). Rapid peptide mapping by reversed-phase liquid chromatography on nonporous silica with online electrospray time of flight mass spectrometry. Anal. Chem. 69(19), 3973-3978. 

Buchanan, N. S., Hamler, R. L., Leopold, P. E., Miller, F. R., Lubman, D. M. (2005). Mass mapping of cancer cell lysates using two-dimensional liquid separations, electrospray-time of flight-mass spectrometry, and automated data processing. Electrophoresis 26(1), 248-256. Buick, R. N., Pullano, R., Trent, J. M. (1985). Comparative properties of 5 human ovarian adenocarcinoma celllines. Cancer Res. 45(8), 3668-3676. 

M. A. Tito, K. Tars, K. Valegard, J. Hajdu, and C. V. Robinson. Electrospray Time-of-Flight Mass Spectrometry of the Intact MS2 Virus Capsid. J. Am. Chem. Soc., 122(2000) 3550-3551. 

Neusiiss, C., Demelbauer, U., and Pelzing, M. (2005). Glycoform characterization of intact erythropoietin by capillary electrophoresis — electrospray-time of flight-mass spectrometry. Electrophoresis 26, 1442—1450. 

Fazar, I. M., Ramsey, R. S., and Ramsey, J. M. (2001). On-chip proteolytic digestion and analysis using wrong-way-round electrospray time-of-flight mass spectrometry. Anal. Chem. 73, 1733-1739. 

Tito, M. A., Tars, K., Valegard, K., Hadju, J., Robinson, C. V Electrospray time-of-flight mass spectrometry of the intact MS2 virus capsid. J Am Chem Soc 2000, 122, 3550-3551. 

Wang, L.T., Amphlett, G., Lambert, J.M. et al. (2005) Structural characterization of a recombinant monoclonal antibody by electrospray time-of-flight mass spectrometry. Pharm. Res., 22, 1338 -9. 

The required instrument hardware was only recently commercialized this may explain why the IPC literature concerning U-HPLC is very limited and contemporary. For example, ion-pair U-HPLC coupled with electrospray time-of-flight mass spectrometry played a crucial role in development of rapid and sensitive analytical... 

Korir, A.K. et al. Ultraperformance ion-pair liquid chromatography coupled to electrospray time-of-flight mass spectrometry for compositional profihng and quantification of heparin and heparan sulfate. Ana/. Chem. 2008, 80, 1297-1306. 

In recent years there has been a growing interest in the use of electrospray ionization-mass spectrometry (ESI-MS) either as a stand-alone technique, or following an analytical separation step like CE, to study and measure a wide variety of compounds in complex samples such us foods (Simo et al. 2005). ESI provides an effective means for ionising from large (e.g., proteins, peptides, carbohydrates) to small (e.g., amino acids, amines) analytes directly from solution prior to their MS analysis without a previous derivatization step. Santos et al. (2004) proposed the use of CE-ESI-MS for the separation and quantification of nine biogenic amines in white and red wines. More recently, the possibilities of two different CE-MS set-ups, namely, capillary electrophoresis-electrospray-ion trap mass spectrometry (CE-IT-MS) and capillary electrophoresis-electrospray-time of flight mass spectrometry (CE-TOE-MS) to analyze directly biogenic amines in wine samples without any previous treatment has been studied (Simo et al. 2008). 

Lazar IM, Lee ED, Rockwood AL, Lee ML. General Considerations for optimizing a capillary elec-trophoresis-electrospray time-of-flight mass spectrometry system, J Chromatogr A 1998 829 279-88. 

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. 

Garces-Claver, A., Amedo-Andres, M. S., Abadia, J., Gil-Ortega, R., and Alvarez-Femandez, A. 2006. Determination of capsaicin and dihydrocapsaicin in capsicum fruits by liquid chromatography-electrospray/time-of-flight mass spectrometry. J. Agric. Food Chem. 54 9303-9311. 

Chen J, Zhao H, Wang X, Lee FS-C, Yang H, Zheng L (2008) Analysis of major alkaloids in Rhizoma coptidis by capillary electrophoiesis-electrospray-time of flight mass spectrometry with different background electrolytes. Electrophoresis 29 2135-2147. doi 10.1002/elps.200700797... 

Truebenbach, C.S. Houalla, M. Hercules, D.M. Characterization of Isopoly Metal Oxyanions Using Electrospray Time-of-Flight Mass Spectrometry. J. Mass Spectrom, 2000, 35, 1121-1127. 

Kozlovski, V Brusov, V Sulimenkov, L Rikhtelev, A. Dodovov, A. Novel experimental arrangement developed for direct fullerene analysis by electrospray time-of-flight mass spectrometry. Rapid Common. Mass Spectrom. 2004, 18, 780-786. 

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