Tandem mass spectroscopy methods

With large proteins, the determination of the primary sequence and post-translational modifications is most efficiently done after digestion with tiypsin or another protease to generate smaller peptides. In this case, the peptides are first separated by HPLC, most commonly RP-HPLC, and the column eluant is directed into the MS. In this hyphenated method, known as LC-MS or liquid chromatography-tandem mass spectroscopy (LC-MS/MS), the individual peptides are analyzed, allowing the identification of post-translational modification sites. In some cases, there are potentially multiple sites in a single peptide that may be modified. 

Lammert, S.A. Cooks, R.G. Pulsed axial activation in the ion trap A new method for performing tandem mass spectroscopy (MS/MS). Rapid Commun. Mass Spectrom. 1992,6, 528-530. 

In such cases, the use of liquid chromatography—tandem mass spectroscopy (LC-MS/MS) is beneficial, since LC-MS/MS provides a sensitive and reproducible measurement compared with conventional methods such as LC—ultraviolet detection or LC—fluorescence detection (Hermo et al., 2008). Furthermore, particularly in the early stages of drug development, is frequently determined by measuring the reduction in the amount of substrate, not the formation of metabolites, because of the difficulty in synthesizing enough amounts of many kinds of metabolites. In such cases, it is necessary to carry out the in vitro reaction so that substrate reduction can be accurately determined. 

It was not possible to use MS for the characterization and identification of chlorophylls and its derivatives until the development of desorption methods (desorption ionization) appropriate for nonvolatile and thermolabile compounds. The mass spectmm of chlorophylls has been obtained using laser desorption [92-94], field desorption [95], plasma desorption [96,97], fast atom bombardment (FAB) [28, 98-101], in-beam electron ionization [102], and electrospray ionization (ESI) [103]. A combination of the techniques of desorption and tandem mass spectroscopy (MS/MS) has also been used for the charactmzation of chlorophylls and derivatives [ 104—107]. The latest research in this field coupled HPLC with MS, using as ionization source FAB [108-110] or atmosphoic pressure chemical ionization (APCI) [111-115]. 

Determination of oxidized amino acids in urine is usually performed by isotope dilution gas chromatography-mass spectrometry (L9). DOPA is estimated by HPLC separation of acid protein hydrolysates with fluorescence detection (excitation 280 nm, emission at 320 nm) (A15). Other methods are based on borate-hydrochloric acid difference spectroscopy (this method suffers interference from tyrosine and tryptophan) (W2), derivatization of DOPA with nitrite and subsequent coulometric determination (W3), and fluorometric detection after derivatization with ethylenediamine (A15). 3-Hydroxylysine is quantitated by HPLC with 9-fluorenylmethyl chloroformate precolumn derivatization (M25) of amino acids obtained by gas-phase hydrolysis of proteins (F21). Other general methods to detect amino acid damage are mass spectometry methods applied to protein hydrolysates, such as tandem mass spectrometry (F6). 

As an instrumental approach to conventional electrophoresis, capillary electrophoresis offers the capability of on-line detection, micropreparative operation and automation (6,8,45-47). In addition, the in tandem connection of capillary electrophoresis to other spectroscopy techniques, such as mass spectrometry, provides high information content on many components of the simple or complex peptide under study. For example, it has been possible to separate and characterize various dynorphins by capillary electrophoresis-mass spectrometry (33). Therefore, the combination of CE-mass spectrometry (CE-MS) provides a valuable analytical tool useful for the fast identification and structural characterization of peptides. Recently, it has been demonstrated that the use of atmospheric pressure ionization using Ion Spray Liquid Chromatography/ Mass Spectrometry is well suited for CE/MS (48). This approach to CE/MS provides a very effective and straightforward method which allow the feasibility of obtaining CE/MS data for peptides from actual biological extracts, i.e., analysis of neuropeptides from equine cerebral spinal fluid (33). 

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