Mass spectrometry solid-phase peptide

S. Beranova-Giorgianni and D. M. Desiderio. Fast Atom Bombardment Mass Spectrometry of Synthetic Peptides. In Methods in Enzymology Solid-Phase Peptide Synthesis, ed. G. B. Fields. Methods in Enzymology 289. Academic Press, San Diego, 1997, 478-499. 

Maux, D. Enjalbal, C. Martinez, J. Aubagnac, J.-L. Combarieu, R. Static Secondary Ion Mass Spectrometry to Monitor Solid-Phase Peptide Synthesis. J. Am. Soc. Mass Spectrom. 2001, 72, 1099-1105. 

Quenched fluorescent substrates (QFS). The QFS listed in Tables 1 and 2 were synthesized by standard solid phase peptide synthesis techniques, and purified by HPLC (see Notes 1 and 2). The identity of each QFS was verified by mass spectrometry. The BK-based QFS (compound no. 26, Table 2), published previously by others (1), was solubiUzed in DMSOiethanol (50 50) at a stock concentration of 2 mg/mL (=1.2 mM), and kept at -20°C (see Note 3). Stock QFS was diluted 1 10 in assay buffer just prior to use. 

Solid phase peptide synthesis of the c-Myc and the Max LZs, characterization by mass spectrometry, purification by reversed-phase HPLC and the formation of the disulfide linked c-Myc-Max heterodimeric LZ have been described elsewhere (19). 

K McMellop, W Davidson, G Hansen, D Freeman, N Pallai. The characterization of crude products from solid-phase peptide synthesis by v-HPLC/fast atom bombardment mass spectrometry. Peptide research 4 40-46, 1991. 

The techniques in solid-phase organic chemistry complement those from solid-phase peptide assembly. Synthesis is performed on resins using anchoring linkages and orthogonal protection schemes. Molecules are cleaved from the solid support, purified (see Chapter 18), and then analyzed by high-performance liquid chromatography or mass spectrometry (see Chapters 19 and 20). Reactions can be performed in a simple apparatus that... 

J. L. Aubaganac, C. Enjalbal, et al.. Application of time-of-flight secondary ion mass spectrometry to in situ monitoring of solid-phase peptide synthesis on the multipin system, J. Mass Spectrom. 33, 1094-1103 (1998). 

Because C-terminal amides have often been reported in insect neuropeptides (8), a 33 residue C-terminal amide consistent with the sequence data was synthesized by solid-phase methods. The peptide was purified by HPLC and its structure confirmed by automated Edman degradation. Californium-252 time-of-flight plasma desorption mass spectrometry provided additional evidence for the structure via a very broad peak for the singly-charged molecular ion at m/z 3902-3906. Because the calculated MW of Hez-PBAN (3899.6, based on the most abundant ion in the isotope cluster) was seen to differ from that observed in the mass spectrum of the isolated native peptide by ca. 32, it was presumed that the native peptide had undergone oxidation of both its methionines to their respective sulfoxides during the course of its isolation and purification. 

The assembly of the p- and y-amino-acid building blocks to peptidic chains was achieved by simply using the established methods of peptide synthesis - in solution [6], on solid phase [11], or in a synthesizer machine [39] also, the so-called native ligation can be applied with p-peptides [54]. Furthermore, the methods of analyzing and studying the structures of a-peptides and natural proteins can mostiy be applied to P-peptides as well (the same is true for y-peptides [51,55-60]). These methods are CD [35,37] and NMR [6, 49] spectroscopy, mass spectrometry [27,35], X-ray analysis [6,21,24,25,36], molecular dynamics (MD) calculations [9,13,18,31,38] and biological investigations [6, 15,20,26,30,41-43,45,46,48]. All of this sounds like routine, but the results are rather spectacular. 

H Zhang, M Stoeckli, PE Andren, RM Caprioli. Combining solid-phase pre-concen-tration, capillary electrophoresis and off-line matrix-assisted laser des-orption/ioniza-tion mass spectrometry intracerebral metabolic processing of peptide E in vivo. J Mass Spectrom 34 377—383, 1999. 

Various molecular and quasi-molecular ions can be formed under MALDI conditions. The formation of protonated analyte (A) molecules, [A -F H]+, is generally most important at least for samples containing slightly basic centres, such as the peptides and proteins, MALDI mass spectrometry of which is known to be most facile and reproducible. Therefore, proton transfer from the electronically excited, neutral or ionized, or protonated matrix species is considered to be crucial in the overall MALDI process . Notably, proton transfer can occur already in the condensed phase, followed by desorption of the preformed ions . However, the generation of the [A -F H]+ ions is believed to take place preferably in the so-called plume , that is, in the energized, short-hved and relatively dense vapour phase generated above the solid matrix upon excitation by the laser pulse. The actual proton donor species (be it one or several) in a given case is still a matter of... 

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