Peptide volatile

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. 

Until 1981, mass spectrometry was limited, generally, to the analysis of volatile, relatively low-molecular-mass samples and was difficult to apply to nonvolatile peptides and proteins without first cutting them chemically into smaller volatile segments. During the past decade, the situation has changed radically with the advent of new ionization techniques and the development of tandem mass spectrometry. Now, the mass spectrometer has a well-deserved place in any laboratory interested in the analysis of peptides and proteins. 

Note Primary amines yield fluorescent chromatogram zones even before the application of reagent 3. Secondary amines do not yield fluorescent derivatives until they have been treated with reagent 3. Hence, the reagent sequence allows the stepwise detection of primary and secondary amines. Taurine is preferred as the essential component of reagent 3 over the multiplicity of other possibilities because it produces intense fluorescence it is also not very volatile and is readily available. Amides and substances with peptide linkages, eg. hippuric acid, are not detected, neither are secondary amines that are volatile at high temperatures. 

The soluble tryptic peptides of 130 mg a chain of Hb-St. Claude were separated on 0.9 x 60 cm columns of Chromobead resin type P (Technlcon Instruments, Dowex 50-X4) at 37°C using the procedure described earlier (16). The method uses a gradient of volatile pyrldlne-acetlc acid buffers of differing molarities and pH as follows first gradient, 666 ml 0.1 M, pH 3.1, and 333 ml 1.0 M, pH 5.0 and second gradient, 166 wl 1.0 M, pH 5.0, and 332 ml 2.0 M, pH 5.0. The amino acid composition of Isolated fragments was determined with a Splnco model 121 automated amino acid analyzer (Beckman Instruments)... 

Schroeder, W. A. "Separation of Peptides by Chromatography on Columns of Dowex 1 with Volatile Developers", In "Methods In Enzymology", p. 214, Vol. XXV, "Enzyme Structure, Part B", C. H. W. Hlrs and S. N. Tlmasheff, Editors, Academic Press, New York, 1972. 

Detection in 2DLC is the same as encountered in one-dimensional HPLC. A variety of detectors are presented in Table 5.2. The choice of detector is dependent on the molecule being detected, the problem being solved, and the separation mode used for the second dimension. If MS detection is utilized, then volatile buffers are typically used in the second-dimension separation. Ultraviolet detection is used for peptides, proteins, and any molecules that contain an appropriate chromophore. Evaporative light scattering detection has become popular for the analysis of polymers and surfactants that do not contain UV chromophores. Refractive index (RI) detection is generally used with size exclusion chromatography for the analysis of polymers. 

In an off-line configuration, a complex peptide mixture from a proteomic sample is loaded onto a SCX column and fractions collected (Fig. 11.1). After the collection of fractions, they are then loaded into an autosampler and analyzed via the traditional RP/ MS/MS approach. Using this system, a variety of buffers and elution conditions may be used (Table 11.1). For example, one may use a volatile salt such as ammonium formate (Adkins et al., 2002 Blonder et al., 2004 Fujii et al., 2004 Yu et al., 2004 Qian et al., 2005a and b) or ammonium acetate (Cutillas et al., 2003 Coldham and Woodward, 2004), collect SCX fractions, lyophilize, resuspend in low acetonitrile and acid, and then directly analyze via RP/MS/MS. In most of the cases, when ammonium acetate or ammonium formate are used, a 20-minute wash period is used to remove the ammonium acetate or ammonium formate prior to the reversed-phase gradient (Table 11.1). However, because fractions are collected and can be buffer exchanged,... 

Analysis using reversed-phase chromatography at low pH was carried out under conditions typically used for 2DLC-MS/MS analysis of peptides. The mobile phases for other chromatographic conditions were also chosen to be compatible with MS detection, including SCX LC, where the peptides were eluted with volatile ammonium formate buffer. 

GC cannot be applied to the analysis of bromocriptine mesilate due to its low volatility and its thermal instability. A procedure according to 29 or 30, which claims excellent identification and quantitation on the basis of well-defined peptide section pyrolysis products, has not yet been attempted. However, GC is very useful determining the residual recrystallization solvent butanone-2. 

FIGURE 15.2 Common protein ionization methods used for MS-based proteomics. Two common ionization technologies are currently available for protein analysis. Top ESI volatilizes and ionizes peptides and proteins in solution. Bottom MALDI uses analytes that are co-crystallized in a matrix composed of organic acid on a solid support. A pulse of ultraviolet laser evaporates the matrix and analyte into gas phase, resulting in generation of single charge ions. 

The use of TFA as a mobile-phase additive in LC-MS can be problematical when using electrospray ionization. In negative ion detection, the high concentration of TFA anion can suppress analyte ionization. In positive ion detection, TFA forms such strong ion pairs with peptides that ejection of peptide pseudo-molecular ions into the gas phase is suppressed. This problem can be alleviated by postcolumn addition of a weaker, less volatile acid such as propionic acid.14 This TFA fix allows TFA to be used with electrospray sources interfaced with quadrupole MS systems. A more convenient solution to the TFA problem in LC-MS is to simply replace TFA with acetic or formic acid. Several reversed-phase columns are commercially available that have sufficient phase coverage and reduced levels of active silanols such that they provide satisfactory peptide peak shapes using the weaker organic acid additives.15... 

Direct injection API-Electrospray MS is capable of analyzing much larger and less volatile substances than either EI/MS or CI/MS. As a result, this method is often used to provide structural information on peptides, proteins, and polymers derived from both natural and synthetic processes it is also useful in the analysis of many natural compounds including molecules such as saponins and flavonol glycosides, derived from plants. When using direct injection API-electrospray, partial purification and EC preparation are performed elsewhere and a collected fraction is dissolved in an appropriate solvent and injected as a bolus into the mass spectrometer (flow or direct injection or syringe infusion). This has an advantage, as the mass... 

---