Liquid chromatography-mass spectrometry peptide determination

The molecular mass of protein and peptide samples was determined by electrospray ionization mass spectrometry using a Perkin-Elmer Sciex API 100 mass spectrometer. The sample was introduced either by infusion or by on-line liquid chromatography/mass spectrometry (LC/MS) using a splitter. The data were obtained by scanning from 450 to 2000 Da with a scan time of 5 s and a step size of 0.25 Da with 1.0 ms dwell time per mass step. The molecular mass of the sample was obtained using the software provided by the instrument manufacturer. 

See a/so Clinical Analysis Sample Handling. Gas Chromatography Mass Spectrometry. Liquid Chromatography Liquid Chromatography-Mass Spectrometry. Mass Spectrometry Ionization Methods Ovenriew Atmospheric Pressure Ionization Techniques Time-of-Flight Selected Ion Monitoring Stable Isotope Ratio. Peptides. Proteins Traditional Methods of quence Determination. Quality Assurance Internal Standards. 

See also-. Electrophoresis Isoelectric Focusing Polyacrylamide Gels Two-Dimensional Gels Proteins. Liquid Chromatography Liquid Chromatography-Mass Spectrometry Biotechnology Applications. Mass Spectrometry Ionization Methods Overview Electrospray Matrix-Assisted Laser Desorption/lonization Multidimensional Peptides and Proteins. Proteins Traditional Methods of Sequence Determination. 

Fig. 9.2 Relative concentrations of P450 in human liver microsomes. a P450s in 60 liver samples were estimated using immunochemical methods (electrophoresis/immunoblot-ting) [52]. Because of cross-reactivity, the individual P450s in subfamilies are not distinguished. The unknown fraction is the difference between the sum of the immunochemi-cally determined forms and the total amount, calculated from Fe -CO versus Fe " difference spectroscopy [53]. b-d Estimates were made using liquid chromatography-mass spectrometry (LC-MS) proteomic analysis with heavy-atom peptides, b Results of an analysis of 50 pooled human liver samples (XenoTech, HLM610 preparation) [54]. c Results reported in the same reference as Part 5 [54] as means from analysis often individual human samples, d Analysis of a pooled set of 23 human liver samples by another laboratory [55]...

Liquid chromatography-mass spectrometry (LC-MS) is an extremely power tool for the analysis of peptides, providing not only information on the purity of the product but also coMormation of structures. The s)rstem typically consists of a microbore HPLC system coupled to an electrospray mass spectrometer. Using such a system the composition of a crude peptide mixture can be quickly determined and by-products identified, enabling synthetic protocols to be rapidly optimized. 

See also Capillary Electrophoresis Overview. Chir-optical Analysis. Liquid Chromatography Column Technology Mobile Phase Selection Reversed Phase Instrumentation Amino Acids. Mass Spectrometry Peptides and Proteins. Nuclear Magnetic Resonance Spectroscopy Techniques Nuclear Overhauser Effect. Proteins Traditional Methods of Sequence Determination Foods. 

Figure 5.11. Generic approaches to identify interacting proteins within complexes. The complex is isolated from cells by affinity purification using a tag sequence attached to a protein known to be in the complex. Alternatively, the complex can be immunprecipitated with an antibody to one of the proteins in the complex. The proteins are resolved by polyacrylamide gel electrophoresis, proteolyzed, and the mass of the resulting peptides is determined by mass spectrometry. Alternatively, the proteins can be proteolyzed and the resulting peptides resolved by liquid chromatography. The peptide masses are then determined by mass spectrometry and used for database searching to identify the component proteins.

Reversed-phase HPLC is widely utilized to generate a peptide map from digested protein, and the MS online method provides rapid identification of the molecular mass of peptides. The HPLC-MS-FAB online system is a sensitive and precise method for low-MW peptides (<3000 Da) even picomol quantities can be detected. However, as the MW of the analytes increases, the ionization of peptides becomes more difficult and decreases the sensibility of the FAB-MS (112). Electrospray ionization (ESI-MS) was found to be an efficient method for the determination of molecular masses up to 200,000 Da of labile biomolecules, with a precision of better than 0.1%. Molecular weights of peptide standards and an extensive hydrolysate of whey protein were determined by the HPLC-MS-FAB online system and supported by MALDI-TOF (112). Furthermore, HPLC-MS-FAB results were compared with those of Fast Performance Liquid Chro-motography (FPLC) analysis. Mass spectrometry coupled with multidimensional automated chromatography for peptide mapping has also been developed (9f,l 12a). 

Liquid chromatography thermospray—mass spectrometry has been utilized to determine the identity and purity of taxol, solvent-induced degradation of cloxacillin [129], benzodiazepines, and ethanolamine-type antihistamines [130]. Electrospray LC-MS has been used for peptide mapping of the human growth hormone [131]. 

The purity of protected amino acids is especially important for the synthesis of longer peptides. Standard techniques such as melting point determination, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and optical rotation are effective means of characterization. The optical purity can also be evaluated by high-performance liquid chromatography (HPLC) after derivatization with Marfey s reagent [216,217]. The advanced Marfey method refers to analysis by mass spectrometry after derivatization with Marfey s reagent [218-221]. Purification of side-chain protected amino acids by recrystallization is usually sufficient. 

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